bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021–09–19
twenty-one papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit
  1. Mapping the evolving landscape of super-enhancers during cell differentiation.
  2. CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.
  3. SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape.
  4. FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.
  5. Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma.
  6. Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.
  7. STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.
  8. Structure of an inactive RNA polymerase II dimer.
  9. Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species.
  10. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity.
  11. UTX condensation underlies its tumour-suppressive activity.
  12. Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression.
  13. DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.
  14. Genetic variations of DNA bindings of FOXA1 and co-factors in breast cancer susceptibility.
  15. A novel form of bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis.
  16. Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer.
  17. TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer.
  18. Nrf2 activation does not affect adenoma development in a mouse model of colorectal cancer.
  19. Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.
  20. Developmental chromatin programs determine oncogenic competence in melanoma.
  21. Activation of gene expression by detergent-like protein domains.
  1. Genome Biol. 2021 Sep 15. 22(1): 269
    Mapping the evolving landscape of super-enhancers during cell differentiation.
    Yan Kai, Bin E Li, Ming Zhu, Grace Y Li, Fei Chen, Yingli Han, Hye Ji Cha, Stuart H Orkin, Wenqing Cai, Jialiang Huang, Guo-Cheng Yuan.
       BACKGROUND: Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure.
    RESULTS: Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes.
    CONCLUSIONS: In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers.
    Keywords:  3D genome; Differentiation; Dynamics; Enhancer; Hierarchy; Super-enhancers
    DOI:  https://doi.org/10.1186/s13059-021-02485-x
  2. Nat Commun. 2021 Sep 13. 12(1): 5415
    CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.
    Martin Franke, Elisa De la Calle-Mustienes, Ana Neto, María Almuedo-Castillo, Ibai Irastorza-Azcarate, Rafael D Acemel, Juan J Tena, José M Santos-Pereira, José L Gómez-Skarmeta.
      Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.
    DOI:  https://doi.org/10.1038/s41467-021-25604-5
  3. Mol Cell. 2021 Sep 10. pii: S1097-2765(21)00696-1. [Epub ahead of print]
    SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape.
    Shibin Hu, Linna Peng, Congling Xu, Zhenning Wang, Aixia Song, Fei Xavier Chen.
      Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.
    Keywords:  INTAC; Integrator; PP2A; Pol II stability; RNA polymerase II; SPT5; elongation; enhancer; pausing; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.029
  4. Nucleic Acids Res. 2021 Sep 17. pii: gkab813. [Epub ahead of print]
    FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.
    Linxuan Zhao, Pengwei Xing, Vamsi Krishna Polavarapu, Miao Zhao, Blanca Valero-Martínez, Yonglong Dang, Nagaprathyusha Maturi, Lucy Mathot, Inês Neves, Irem Yildirim, Fredrik Johansson Swartling, Tobias Sjöblom, Lene Uhrbom, Xingqi Chen.
      The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.
    DOI:  https://doi.org/10.1093/nar/gkab813
  5. Nat Commun. 2021 Sep 14. 12(1): 5450
    Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma.
    Jaime Alvarez-Benayas, Nikolaos Trasanidis, Alexia Katsarou, Kanagaraju Ponnusamy, Aristeidis Chaidos, Philippa C May, Xiaolin Xiao, Marco Bua, Maria Atta, Irene A G Roberts, Holger W Auner, Evdoxia Hatjiharissi, Maria Papaioannou, Valentina S Caputo, Ian M Sudbery, Anastasios Karadimitris.
      Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.
    DOI:  https://doi.org/10.1038/s41467-021-25704-2
  6. Proc Natl Acad Sci U S A. 2021 Sep 21. pii: e2109475118. [Epub ahead of print]118(38):
    Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.
    Masaki Kinoshita, Meng Amy Li, Michael Barber, William Mansfield, Sabine Dietmann, Austin Smith.
      Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2 Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2 However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.
    Keywords:  DNA methylation; embryonic stem cells; pluripotency
    DOI:  https://doi.org/10.1073/pnas.2109475118
  7. Mol Cell. 2021 Sep 07. pii: S1097-2765(21)00686-9. [Epub ahead of print]
    STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.
    Joshua T Zimmer, Nicolle A Rosa-Mercado, Daniele Canzio, Joan A Steitz, Matthew D Simon.
      Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.
    Keywords:  STL-seq; TimeLapse; premature termination; promoter-proximal pausing; release into elongation; scRNA
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.019
  8. Nucleic Acids Res. 2021 Sep 16. pii: gkab783. [Epub ahead of print]
    Structure of an inactive RNA polymerase II dimer.
    Shintaro Aibara, Christian Dienemann, Patrick Cramer.
      Eukaryotic gene transcription is carried out by three RNA polymerases: Pol I, Pol II and Pol III. Although it has long been known that Pol I can form homodimers, it is unclear whether and how the two other RNA polymerases dimerize. Here we present the cryo-electron microscopy (cryo-EM) structure of a mammalian Pol II dimer at 3.5 Å resolution. The structure differs from the Pol I dimer and reveals that one Pol II copy uses its RPB4-RPB7 stalk to penetrate the active centre cleft of the other copy, and vice versa, giving rise to a molecular handshake. The polymerase clamp domain is displaced and mobile, and the RPB7 oligonucleotide-binding fold mimics the DNA-RNA hybrid that occupies the cleft during active transcription. The Pol II dimer is incompatible with nucleic acid binding as required for transcription and may represent an inactive storage form of the polymerase.
    DOI:  https://doi.org/10.1093/nar/gkab783
  9. Elife. 2021 Sep 13. pii: e67964. [Epub ahead of print]10
    Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species.
    Natalia Petrenko, Kevin Struhl.
      The preinitation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TBP and associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.
    Keywords:  D. melanogaster; S. cerevisiae; S. pombe; chromosomes; gene expression; genetics; genomics; human; mouse
    DOI:  https://doi.org/10.7554/eLife.67964
  10. Nat Commun. 2021 Sep 17. 12(1): 5520
    Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity.
    Casey G Langdon, Katherine E Gadek, Matthew R Garcia, Myron K Evans, Kristin B Reed, Madeline Bush, Jason A Hanna, Catherine J Drummond, Matthew C Maguire, Patrick J Leavey, David Finkelstein, Hongjian Jin, Patrick A Schreiner, Jerold E Rehg, Mark E Hatley.
      PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.
    DOI:  https://doi.org/10.1038/s41467-021-25829-4
  11. Nature. 2021 Sep 15.
    UTX condensation underlies its tumour-suppressive activity.
    Bi Shi, Wei Li, Yansu Song, Zhenjia Wang, Rui Ju, Aleksandra Ulman, Jing Hu, Francesco Palomba, Yanfang Zhao, John Philip Le, William Jarrard, David Dimoff, Michelle A Digman, Enrico Gratton, Chongzhi Zang, Hao Jiang.
      UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity of MLL4. Moreover, UTX regulates genome-wide histone modifications and high-order chromatin interactions in a condensation-dependent manner. We also found that UTY, the Y chromosome homologue of UTX with weaker tumour-suppressive activity, forms condensates with reduced molecular dynamics. These studies demonstrate a crucial biological function of liquid condensates with proper material states in enabling the tumour-suppressive activity of a chromatin regulator.
    DOI:  https://doi.org/10.1038/s41586-021-03903-7
  12. Nat Commun. 2021 Sep 13. 12(1): 5402
    Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression.
    Mathieu F Bakhoum, Jasmine H Francis, Albert Agustinus, Ethan M Earlie, Melody Di Bona, David H Abramson, Mercedes Duran, Ignas Masilionis, Elsa Molina, Alexander N Shoushtari, Michael H Goldbaum, Paul S Mischel, Samuel F Bakhoum, Ashley M Laughney.
      Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-021-25529-z
  13. Nat Commun. 2021 Sep 13. 12(1): 5406
    DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.
    Rajbir Nath Batra, Aviezer Lifshitz, Ana Tufegdzic Vidakovic, Suet-Feung Chin, Ankita Sati-Batra, Stephen-John Sammut, Elena Provenzano, H Raza Ali, Ali Dariush, Alejandra Bruna, Leigh Murphy, Arnie Purushotham, Ian Ellis, Andrew Green, Francine E Garrett-Bakelman, Chris Mason, Ari Melnick, Samuel A J R Aparicio, Oscar M Rueda, Amos Tanay, Carlos Caldas.
      DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.
    DOI:  https://doi.org/10.1038/s41467-021-25661-w
  14. Nat Commun. 2021 Sep 13. 12(1): 5318
    Genetic variations of DNA bindings of FOXA1 and co-factors in breast cancer susceptibility.
    Wanqing Wen, Zhishan Chen, Jiandong Bao, Quan Long, Xiao-Ou Shu, Wei Zheng, Xingyi Guo.
      Identifying transcription factors (TFs) whose DNA bindings are altered by genetic variants that regulate susceptibility genes is imperative to understand transcriptional dysregulation in disease etiology. Here, we develop a statistical framework to analyze extensive ChIP-seq and GWAS data and identify 22 breast cancer risk-associated TFs. We find that, by analyzing genetic variations of TF-DNA bindings, the interaction of FOXA1 with co-factors such as ESR1 and E2F1, and the interaction of TFs with chromatin features (i.e., enhancers) play a key role in breast cancer susceptibility. Using genetic variants occupied by the 22 TFs, transcriptome-wide association analyses identify 52 previously unreported breast cancer susceptibility genes, including seven with evidence of essentiality from functional screens in breast relevant cell lines. We show that FOXA1 and co-factors form a core TF-transcriptional network regulating the susceptibility genes. Our findings provide additional insights into genetic variations of TF-DNA bindings (particularly for FOXA1) underlying breast cancer susceptibility.
    DOI:  https://doi.org/10.1038/s41467-021-25670-9
  15. Elife. 2021 Sep 15. pii: e69508. [Epub ahead of print]10
    A novel form of bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis.
    Kangmei Zhao, Deze Kong, Benjamin Jin, Christina D Smolke, Seung Yon Rhee.
      Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, H3K27me3 (repression) and H3K18ac (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27m3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name a kairostat, plays an important role controlling the timely induction of gene expression upon stimuli in plants.
    Keywords:  A. thaliana; plant biology
    DOI:  https://doi.org/10.7554/eLife.69508
  16. Genes Dev. 2021 Sep 16.
    Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer.
    Yuxiao Zhou, Siyuan Xu, Mo Zhang, Qiang Wu.
      Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.
    Keywords:  3D chromatin structure; R-loop formation; RNase H1; SPT6; enhancer RNA PEARL; long-distance enhancer-promoter looping; protocadherin gene expression; superenhancer
    DOI:  https://doi.org/10.1101/gad.348621.121
  17. Proc Natl Acad Sci U S A. 2021 Sep 21. pii: e2102423118. [Epub ahead of print]118(38):
    TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer.
    Neeraj Agarwal, Sebastien Rinaldetti, Bassem B Cheikh, Qiong Zhou, Evan P Hass, Robert T Jones, Molishree Joshi, Daniel V LaBarbera, Simon R V Knott, Thomas R Cech, Dan Theodorescu.
      Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.
    Keywords:  CRISPR-Cas9 KnockIn; Kinome KO screening; hTERT; promoter mutation
    DOI:  https://doi.org/10.1073/pnas.2102423118
  18. Commun Biol. 2021 Sep 15. 4(1): 1081
    Nrf2 activation does not affect adenoma development in a mouse model of colorectal cancer.
    Elena V Knatko, Cecilia Castro, Maureen Higgins, Ying Zhang, Tadashi Honda, Colin J Henderson, C Roland Wolf, Julian L Griffin, Albena T Dinkova-Kostova.
      Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism. Nrf2 activation is protective in models of human disease and has benefits in clinical trials. Consequently, the Keap1/Nrf2 protein complex is a drug target. However, in cancer Nrf2 plays a dual role, raising concerns that Nrf2 activators may promote growth of early neoplasms. To address this concern, we examined the role of Nrf2 in development of colorectal adenomas by employing genetic, pharmacological, and metabolomic approaches. We found that colorectal adenomas that form in Gstp-/-: ApcMin/+ mice are characterized by altered one-carbon metabolism and that genetic activation, but not disruption of Nrf2, enhances these metabolic alterations. However, this enhancement is modest compared to the magnitude of metabolic differences between tumor and peri-tumoral tissues, suggesting that the metabolic changes conferred by Nrf2 activation may have little contribution to the early stages of carcinogenesis. Indeed, neither genetic (by Keap1 knockdown) nor pharmacological Nrf2 activation, nor its disruption, affected colorectal adenoma formation in this model. We conclude that pharmacological Nrf2 activation is unlikely to impact the early stages of development of colorectal cancer.
    DOI:  https://doi.org/10.1038/s42003-021-02552-w
  19. Gut. 2021 Sep 11. pii: gutjnl-2021-325405. [Epub ahead of print]
    Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.
    Steffie Revia, Agnieszka Seretny, Lena Wendler, Ana Banito, Christoph Eckert, Kersten Breuer, Anand Mayakonda, Pavlo Lutsik, Matthias Evert, Silvia Ribback, Suchira Gallage, Ismaiel Chikh Bakri, Kai Breuhahn, Peter Schirmacher, Stefan Heinrich, Matthias M Gaida, Mathias Heikenwälder, Diego F Calvisi, Christoph Plass, Scott W Lowe, Darjus F Tschaharganeh.
       OBJECTIVE: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer.
    DESIGN: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted.
    RESULTS: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition.
    CONCLUSION: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.
    Keywords:  gastrointestinal cancer; hepatobiliary cancer; hepatocellular carcinoma; molecular carcinogenesis
    DOI:  https://doi.org/10.1136/gutjnl-2021-325405
  20. Science. 2021 09 03. 373(6559): eabc1048
    Developmental chromatin programs determine oncogenic competence in melanoma.
    Arianna Baggiolini, Scott J Callahan, Emily Montal, Joshua M Weiss, Tuan Trieu, Mohita M Tagore, Sam E Tischfield, Ryan M Walsh, Shruthy Suresh, Yujie Fan, Nathaniel R Campbell, Sarah C Perlee, Nathalie Saurat, Miranda V Hunter, Theresa Simon-Vermot, Ting-Hsiang Huang, Yilun Ma, Travis Hollmann, Satish K Tickoo, Barry S Taylor, Ekta Khurana, Richard P Koche, Lorenz Studer, Richard M White.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/science.abc1048
  21. iScience. 2021 Sep 24. 24(9): 103017
    Activation of gene expression by detergent-like protein domains.
    Bradley K Broyles, Andrew T Gutierrez, Theodore P Maris, Daniel A Coil, Thomas M Wagner, Xiao Wang, Daisuke Kihara, Caleb A Class, Alexandre M Erkine.
      The mechanisms by which transcriptional activation domains (tADs) initiate eukaryotic gene expression have been an enigma for decades because most tADs lack specificity in sequence, structure, and interactions with targets. Machine learning analysis of data sets of tAD sequences generated in vivo elucidated several functionality rules: the functional tAD sequences should (i) be devoid of or depleted with basic amino acid residues, (ii) be enriched with aromatic and acidic residues, (iii) be with aromatic residues localized mostly near the terminus of the sequence, and acidic residues localized more internally within a span of 20-30 amino acids, (iv) be with both aromatic and acidic residues preferably spread out in the sequence and not clustered, and (v) not be separated by occasional basic residues. These and other more subtle rules are not absolute, reflecting absence of a tAD consensus sequence, enormous variability, and consistent with surfactant-like tAD biochemical properties. The findings are compatible with the paradigm-shifting nucleosome detergent mechanism of gene expression activation, contributing to the development of the liquid-liquid phase separation model and the biochemistry of near-stochastic functional allosteric interactions.
    Keywords:  Bioinformatics; Biological sciences; Computational bioinformatics; IDR; Molecular biology; chromatin; gene activation; machine learning; neural network; nucleosome; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.isci.2021.103017
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