bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2022‒10‒23
25 papers selected by
Connor Rogerson
University of Cambridge

  1. Nucleic Acids Res. 2022 Oct 16. pii: gkac881. [Epub ahead of print]
      Cancer is a disease of gene dysregulation, where cells acquire somatic and epigenetic alterations that drive aberrant cellular signaling. These alterations adversely impact transcriptional programs and cause profound changes in gene expression. Interpreting somatic alterations within context-specific transcriptional programs will facilitate personalized therapeutic decisions but is a monumental task. Toward this goal, we develop a partially interpretable neural network model called Chromatin-informed Inference of Transcriptional Regulators Using Self-attention mechanism (CITRUS). CITRUS models the impact of somatic alterations on transcription factors and downstream transcriptional programs. Our approach employs a self-attention mechanism to model the contextual impact of somatic alterations. Furthermore, CITRUS uses a layer of hidden nodes to explicitly represent the state of transcription factors (TFs) to learn the relationships between TFs and their target genes based on TF binding motifs in the open chromatin regions of tumor samples. We apply CITRUS to genomic, transcriptomic, and epigenomic data from 17 cancer types profiled by The Cancer Genome Atlas. CITRUS predicts patient-specific TF activities and reveals transcriptional program variations between and within tumor types. We show that CITRUS yields biological insights into delineating TFs associated with somatic alterations in individual tumors. Thus, CITRUS is a promising tool for precision oncology.
  2. Genome Biol. 2022 Oct 17. 23(1): 221
      BACKGROUND: We and others have suggested that pioneer activity - a transcription factor's (TF's) ability to bind and open inaccessible loci - is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci.RESULTS: Here, we present a quantitative in vivo measure of pioneer activity that captures the relative difference in a TF's ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure the binding of doxycycline-inducible TFs. For each location across the genome, we determine the concentration of doxycycline required for a TF to reach half-maximal occupancy; lower concentrations reflect higher affinity. We propose that the relative difference in a TF's affinity between ATAC-seq labeled accessible and inaccessible binding sites is a measure of its pioneer activity. We estimate binding affinities at tens of thousands of genomic loci for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has stronger pioneer activity than FOXA1. We show that both FOXA1 and HNF4A display higher binding affinity at inaccessible sites with more copies of their respective motifs. The quantitative analysis of binding suggests different modes of binding for FOXA1, including an anti-cooperative mode of binding at certain accessible loci.
    CONCLUSIONS: Our results suggest that relative binding affinities are reasonable measures of pioneer activity and support the model wherein most TFs have some degree of context-dependent pioneer activity.
    Keywords:  FOXA1; Genomics; HNF4A; Pioneer activity; Pioneer factor; Transcription factor binding
  3. EMBO Rep. 2022 Oct 17. e54720
      Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher-order chromatin organization and transcription regulation. However, it is still not clear how long-range contacts contribute to transcriptional regulation. Here, we show that relative-of-WOC (ROW) is essential for the long-range transcription regulation mediated by the boundary element-associated factor of 32kD (BEAF-32). We find that ROW physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein (BEAF-32). These proteins interact at TAD boundaries where ROW, through its AT-hook motifs, binds AT-rich sequences flanked by BEAF-32-binding sites and motifs. Knockdown of row downregulates genes that are long-range targets of BEAF-32 and bound indirectly by ROW (without binding motif). Analyses of high-throughput chromosome conformation capture (Hi-C) data reveal long-range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, our results show cooperation between BEAF-32 and the ROW complex, including HP1 proteins, to regulate the transcription of developmental and inducible genes through long-range interactions.
    Keywords:   Drosophila ; gene regulation; heterochromatin proteins; insulator proteins; topological associated domains (TADs)
  4. Circ Res. 2022 Oct 20.
      BACKGROUND: The pioneer transcription factor GATA4 is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression.METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays.
    RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 and ETS1 motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity.
    CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF (transcription factor) GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.
    Keywords:  animals; chromatin; gene expression; genomics; immunoprecipitation
  5. Cell Rep. 2022 Oct 18. pii: S2211-1247(22)01351-1. [Epub ahead of print]41(3): 111501
      The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.
    Keywords:  CP: Developmental biology; CP: Molecular biology; CTCF; Hi-C; blastocyst; genome structure; metabolism; morula; preimplantation development
  6. Nat Commun. 2022 Oct 20. 13(1): 6230
      TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.
  7. Development. 2022 Oct 20. pii: dev.200940. [Epub ahead of print]
      Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called "shadow enhancers," which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, however the biological significance of this remains unclear. In this study we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer, and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation, and RNA Polymerase II loading rates. Additionally, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. While activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that can be explained by the differences in transcriptional kinetics measured.
    Keywords:  MS2 live imaging; Morphogen gradient; Shadow enhancers; Transcriptional kinetics
  8. Cell Rep. 2022 Oct 18. pii: S2211-1247(22)01341-9. [Epub ahead of print]41(3): 111491
      Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.
    Keywords:  CP: Molecular biology; DNA loop extrusion; RNA polymerase; SMC; cohesin; condensin; dCas9; mechanism; nucleosomes; roadblocks; topology
  9. Genome Biol. 2022 Oct 17. 23(1): 217
      BACKGROUND: The early embryonic divisions of many organisms, including fish, flies, and frogs, are characterized by a very rapid S-phase caused by high rates of replication initiation. In somatic cells, S-phase is much longer due to both a reduction in the total number of initiation events and the imposition of a temporal order of origin activation. The physiological importance of changes in the rate and timing of replication initiation in S-phase remains unclear.RESULTS: Here we assess the importance of the temporal control of replication initiation using a conditional system in budding yeast to drive the early replication of the majority of origins in a single cell cycle. We show that global early replication disrupts the expression of over a quarter of all genes. By deleting individual origins, we show that delaying replication is sufficient to restore normal gene expression, directly implicating origin firing control in this regulation. Global early replication disrupts nucleosome positioning and transcription factor binding during S-phase, suggesting that the rate of S-phase is important to regulate the chromatin landscape.
    CONCLUSIONS: Together, these data provide new insight into the role of the temporal control of origin firing during S-phase for coordinating replication, gene expression, and chromatin establishment as occurs in the early embryo.
  10. NAR Genom Bioinform. 2022 Dec;4(4): lqac075
      Score-based motif enrichment analysis (MEA) is typically applied to regulatory DNA to infer transcription factors (TFs) that may modulate transcription and chromatin state in different conditions. Most MEA methods determine motif enrichment independent of motif position within a sequence, even when those sequences harbor anchor points that motifs and their bound TFs may functionally interact with in a distance-dependent fashion, such as other TF binding motifs, transcription start sites (TSS), sequencing assay cleavage sites, or other biologically meaningful features. We developed motif enrichment positional profiling (MEPP), a novel MEA method that outputs a positional enrichment profile of a given TF's binding motif relative to key anchor points (e.g. transcription start sites, or other motifs) within the analyzed sequences while accounting for lower-order nucleotide bias. Using transcription initiation and TF binding as test cases, we demonstrate MEPP's utility in determining the sequence positions where motif presence correlates with measures of biological activity, inferring positional dependencies of binding site function. We demonstrate how MEPP can be applied to interpretation and hypothesis generation from experiments that quantify transcription initiation, chromatin structure, or TF binding measurements. MEPP is available for download from
  11. Nat Genet. 2022 Oct 17.
      Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.
  12. Cell Syst. 2022 Oct 19. pii: S2405-4712(22)00395-7. [Epub ahead of print]13(10): 798-807.e6
      Single-cell Hi-C (scHi-C) technologies can probe three-dimensional (3D) genome structures in individual cells. However, existing scHi-C analysis methods are hindered by the data quality and complex 3D genome patterns. The lack of computational scalability and interpretability poses further challenges for large-scale analysis. Here, we introduce Fast-Higashi, an ultrafast and interpretable method based on tensor decomposition and partial random walk with restart, enabling joint identification of cell identities and chromatin meta-interactions from sparse scHi-C data. Extensive evaluations demonstrate the advantage of Fast-Higashi over existing methods, leading to improved delineation of rare cell types and continuous developmental trajectories. Fast-Higashi can directly identify 3D genome features that define distinct cell types and help elucidate cell-type-specific connections between genome structure and function. Moreover, Fast-Higashi can generalize to incorporate other single-cell omics data. Fast-Higashi provides a highly efficient and interpretable scHi-C analysis solution that is applicable to a broad range of biological contexts.
    Keywords:  chromatin meta-interaction; machine learning; single-cell 3D genome; single-cell Hi-C; tensor decomposition
  13. Elife. 2022 Oct 20. pii: e81400. [Epub ahead of print]11
      The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1 Å-resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the HAT and TINTIN modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1 and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4 and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggests a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.
    Keywords:  S. cerevisiae; molecular biophysics; structural biology
  14. Cell Rep. 2022 Oct 18. pii: S2211-1247(22)01342-0. [Epub ahead of print]41(3): 111492
      Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation. To investigate the role of supercoiling-mediated feedback within multi-gene systems, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical inter-gene coupling. We find that gene syntax-the relative ordering and orientation of genes-defines the expression profiles, variance, burst dynamics, and inter-gene correlation of two-gene systems. Furthermore, supercoiling can enhance or weaken biochemical regulation. Our results suggest that supercoiling couples behavior between neighboring genes, providing a regulatory mechanism that tunes transcriptional variance in engineered gene networks and explains the behavior of co-localized native circuits.
    Keywords:  CP: Molecular biology; DNA supercoiling; gene circuits; gene expression; noise; stochastic simulations; synthetic biology; transcriptional bursting; transcriptional dynamics
  15. BMC Genomics. 2022 Oct 19. 23(1): 714
      BACKGROUND: Mouse is probably the most important model organism to study mammal biology and human diseases. A better understanding of the mouse genome will help understand the human genome, biology and diseases. However, despite the recent progress, the characterization of the regulatory sequences in the mouse genome is still far from complete, limiting its use to understand the regulatory sequences in the human genome.RESULTS: Here, by integrating binding peaks in ~ 9,000 transcription factor (TF) ChIP-seq datasets that cover 79.9% of the mouse mappable genome using an efficient pipeline, we were able to partition these binding peak-covered genome regions into a cis-regulatory module (CRM) candidate (CRMC) set and a non-CRMC set. The CRMCs contain 912,197 putative CRMs and 38,554,729 TF binding sites (TFBSs) islands, covering 55.5% and 24.4% of the mappable genome, respectively. The CRMCs tend to be under strong evolutionary constraints, indicating that they are likely cis-regulatory; while the non-CRMCs are largely selectively neutral, indicating that they are unlikely cis-regulatory. Based on evolutionary profiles of the genome positions, we further estimated that 63.8% and 27.4% of the mouse genome might code for CRMs and TFBSs, respectively.
    CONCLUSIONS: Validation using experimental data suggests that at least most of the CRMCs are authentic. Thus, this unprecedentedly comprehensive map of CRMs and TFBSs can be a good resource to guide experimental studies of regulatory genomes in mice and humans.
    Keywords:  Cis-regulatory modules; Mouse; Transcription factor binding sites
  16. Sci Rep. 2022 Oct 20. 12(1): 17586
      The transcription factor hepatocyte nuclear factor 1β (HNF-1β) is essential for normal development of the kidney and other epithelial organs. In the developing mouse kidney, HNF-1β is required for the differentiation and patterning of immature nephrons and branching morphogenesis of the ureteric bud (UB). Here, we used ChIP-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to identify genes that are regulated by HNF-1β in embryonic mouse kidneys. ChIP-seq revealed that HNF-1β binds to 8284 sites in chromatin from E14.5 mouse kidneys. Comparison with previous ATAC-seq and histone modification studies showed that HNF-1β binding peaks colocalized with open chromatin and epigenetic marks of transcriptional activation (H3K27 acetylation, H3K4 trimethylation, H3K4 monomethylation), indicating that the binding sites were functional. To investigate the relationship between HNF-1β binding and HNF-1β-dependent gene regulation, RNA-seq was performed on UB cells purified from wild-type and HNF-1β mutant embryonic kidneys. A total of 1632 genes showed reduced expression in HNF-1β-deficient UB cells, and 485 genes contained nearby HNF-1β binding sites indicating that they were directly activated by HNF-1β. Conversely, HNF-1β directly repressed the expression of 526 genes in the UB. Comparison with snATAC-seq analysis of UB-derived cells showed that both HNF-1β-dependent activation and repression correlated with chromatin accessibility. Pathway analysis revealed that HNF-1β binds near 68 axon guidance genes in the developing kidney. RNA-seq analysis showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were activated by HNF-1β, whereas Efna1, Epha3, Epha4, Epha7, Ntn4, Plxna2, Sema3a, Sema4b, Slit3, Srgap1, Unc5c and Unc5d were repressed by HNF-1β. RNAscope in situ hybridization showed that Nrp1, Sema3c, Sema3d, Sema6a, and Slit2 were expressed in wild-type UB and were dysregulated in HNF-1β mutant UB. These studies show that HNF-1β directly regulates the expression of multiple axon guidance genes in the developing mouse kidney. Dysregulation of axon guidance genes may underlie kidney defects in HNF-1β mutant mice.
  17. Nat Commun. 2022 Oct 17. 13(1): 6041
      Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens.
  18. Mol Cell. 2022 Oct 20. pii: S1097-2765(22)00957-1. [Epub ahead of print]82(20): 3840-3855.e8
      The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.
    Keywords:  Bcl2; CAP-independent translation; Hidden Markov Model; N-terminal mass spectrometry; N6-methyladenosine; RNA structure; alternative cleavage and polyadenylation; immunopeptidome; mammalian transcriptome; uncapped RNA
  19. Sci Adv. 2022 Oct 21. 8(42): eabq8297
      Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.
  20. Nat Commun. 2022 Oct 20. 13(1): 6206
      Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.
  21. Nat Commun. 2022 Oct 17. 13(1): 6133
      Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-JUN proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-JUN multisite phosphorylation has remained elusive. Here we show that c-JUN phosphorylation by JNK exhibits defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identify the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis reveals three c-JUN phosphorylation states: unphosphorylated c-JUN recruits the MBD3 repressor, serine63/73 doubly-phosphorylated c-JUN binds to the TCF4 co-activator, whereas the fully phosphorylated form disfavours TCF4 binding attenuating JNK signalling. Thus, c-JUN phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.
  22. Elife. 2022 Oct 21. pii: e57736. [Epub ahead of print]11
      The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.
    Keywords:  chromosomes; gene expression; human
  23. Nucleic Acids Res. 2022 Oct 21. pii: gkac907. [Epub ahead of print]
      Transcription factors (TFs) are proteins that interact with specific DNA sequences to regulate gene expression and play crucial roles in all kinds of biological processes. To keep up with new data and provide a more comprehensive resource for TF research, we updated the Animal Transcription Factor Database (AnimalTFDB) to version 4.0 ( with up-to-date data and functions. We refined the TF family rules and prediction pipeline to predict TFs in genome-wide protein sequences from Ensembl. As a result, we predicted 274 633 TF genes and 150 726 transcription cofactor genes in AnimalTFDB 4.0 in 183 animal genomes, which are 86 more species than AnimalTFDB 3.0. Besides double data volume, we also added the following new annotations and functions to the database: (i) variations (including mutations) on TF genes in various human cancers and other diseases; (ii) predicted post-translational modification sites (including phosphorylation, acetylation, methylation and ubiquitination sites) on TFs in 8 species; (iii) TF regulation in autophagy; (iv) comprehensive TF expression annotation for 38 species; (v) exact and batch search functions allow users to search AnimalTFDB flexibly. AnimalTFDB 4.0 is a useful resource for studying TF and transcription regulation, which contains comprehensive annotation and classification of TFs and transcription cofactors.
  24. Cancer Res. 2022 Oct 17. 82(20): 3668-3670
      Invasive lobular carcinomas (ILC) are the second most common histologic subtype of breast cancer, accounting for up to 15% of cases. ILC is estrogen receptor (ER) positive, yet its biology is distinct from invasive ductal carcinomas (IDC), and retrospective analyses have indicated a poorer outcome with endocrine therapy. In this issue of Cancer Research, Nardone and colleagues investigated the mechanisms of this differential therapy response in ILC, which cannot be solely explained by the genetic profile of these tumors. The authors conducted a thorough examination of the epigenome of ILC compared with IDC in clinical and preclinical models and revealed an alternative chromatin accessibility state in ILC driven by the pioneer factor FOXA1. FOXA1 regulates its own expression in a feed-forward mechanism by binding to an ILC-unique FOXA1 enhancer site. This results in a FOXA1-ER axis that promotes the transcription of genes associated with tumor progression and tamoxifen resistance. Targeting the FOXA1 enhancer region blocks this transcriptional program and inhibits ILC proliferation. These results shed light on a new epigenetic mechanism driving ILC tumor progression and treatment resistance, which may have profound therapeutic implications. See related article by Nardone et al., p. 3673.
  25. Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2209211119
      About one-fourth of recurrent estrogen receptor-positive (ER+) breast cancers lose ER expression, leading to endocrine therapy failure. However, the mechanisms underlying ER loss remain to be fully explored. We now show that 14-3-3τ, up-regulated in ∼60% of breast cancer, drives the conversion of ER+ to ER- and epithelial-to-mesenchymal transition (EMT). We identify ERα36, an isoform of ERα66, as a downstream effector of 14-3-3τ. Overexpression of 14-3-3τ induces ERα36 in xenografts and tumor spheroids. The regulation is further supported by a positive correlation between ERα36 and 14-3-3τ expression in human breast cancers. ERα36 can antagonize ERα66 and inhibit ERα66 expression. Isoform-specific depletion of ERα36 blocks the ER conversion and EMT induced by 14-3-3τ overexpression in tumor spheroids, thus establishing ERα36 as a key mediator in 14-3-3τ-driven ER loss and EMT. ERα36 promoter is repressed by GATA3, which can be phosphorylated by AKT at consensus binding sites for 14-3-3. Upon AKT activation, 14-3-3τ binds phosphorylated GATA3 and facilitates the degradation of GATA3 causing GATA3 to lose transcriptional control over its target genes ERα66 and ERα36. We also demonstrate a role for the collaboration between 14-3-3τ and AKT in ERα36 induction and endocrine therapy resistance by three-dimensional spheroid and tamoxifen treatment models in MCF7 and T47D ER+ breast cancer cells. Thus, the 14-3-3τ-ERα36 regulation provides a previously unrecognized mechanism for ER loss and endocrine therapy failure.
    Keywords:  14-3-3τ; 3D tumor spheroid model; ERα36; GATA3; estrogen receptor