A complex epigenome-splicing crosstalk governs epithelial-to-mesenchymal transition in metastasis and brain development.

Epithelial-to-mesenchymal transition (EMT) renders epithelial cells migratory properties. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here we discovered that a C2H2 zinc finger protein, ZNF827, is strongly induced during various contexts of EMT, including in brain development and breast cancer metastasis, and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodelling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA polymerase II progression and altering the splicing of genes encoding key EMT regulators in cis. Our findings reveal an unprecedented complexity of crosstalk between epigenetic landscape and splicing programme in governing EMT and identify ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Myc Supports Self-Renewal of Basal Cells in the Esophageal Epithelium.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

Measuring Chromatin Accessibility: ATAC-Seq.

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) is a method to investigate the accessibility of chromatin in a genome-wide fashion. In this chapter, we provide a brief history of the chromatin accessibility field followed by a detailed protocol to perform ATAC-Seq assay.

Role of viral suppressors governing asymmetric synergism between tomato-infecting begomoviruses.

Mixed viral infections are common in fields and frequently exacerbate disease severity via synergistic interactions among individual viral genomic components leading to major crop loss. Two predominant species of tomato-infecting begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl Gujarat virus (ToLCGuV), are known to cause severe leaf curl disease of tomato in India. Previously, we have demonstrated asymmetric synergism between these two distinct begomovirus species during mixed infection in solanaceous hosts. In the present study, we have identified the underlying proteins that positively regulate asymmetric synergism and their effect on plant defense machinery. During co-infection, the AC2 and AV2 of ToLCGuV enhanced ToLCNDV DNA accumulation in Nicotiana benthamiana as well as in their natural host, tomato. Furthermore, we found that AC2 and AV2 of ToLCNDV and AV2 of ToLCGuV play a critical role in suppression of post transcriptional gene silencing (PTGS) machinery. Taken together, AC2 and AV2 encoded proteins of ToLCGuV are the crucial viral factors promoting asymmetric synergism with ToLCNDV. KEY POINTS: • Begomoviral suppressors play vital roles in viral synergism. • AC2 and AV2 of ToLCGuV asymmetrically enhance ToLCNDV accumulation. • AC2 and AV2 of ToLCNDV and ToLCGuV AV2 are major PTGS suppressors.

Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation.

Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neurogenesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subsequently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes.

A broad molecular framework of how neural stem cells are specified toward astrocyte fate during brain development has proven elusive. Here we perform comprehensive and integrated transcriptomic and epigenomic analyses to delineate gene regulatory programs that drive the developmental trajectory from mouse embryonic stem cells to astrocytes. We report molecularly distinct phases of astrogliogenesis that exhibit stage- and lineage-specific transcriptomic and epigenetic signatures with unique primed and active chromatin regions, thereby revealing regulatory elements and transcriptional programs underlying astrocyte generation and maturation. By searching for transcription factors that function at these elements, we identified NFIA and ATF3 as drivers of astrocyte differentiation from neural precursor cells while RUNX2 promotes astrocyte maturation. These transcription factors facilitate stage-specific gene expression programs by switching the chromatin state of their target regulatory elements from primed to active. Altogether, these findings provide integrated insights into the genetic and epigenetic mechanisms steering the trajectory of astrogliogenesis.

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.

FBXO32 promotes microenvironment underlying epithelial-mesenchymal transition via CtBP1 during tumour metastasis and brain development.

The set of events that convert adherent epithelial cells into migratory cells are collectively known as epithelial-mesenchymal transition (EMT). EMT is involved during development, for example, in triggering neural crest migration, and in pathogenesis such as metastasis. Here we discover FBXO32, an E3 ubiquitin ligase, to be critical for hallmark gene expression and phenotypic changes underlying EMT. Interestingly, FBXO32 directly ubiquitinates CtBP1, which is required for its stability and nuclear retention. This is essential for epigenetic remodeling and transcriptional induction of CtBP1 target genes, which create a suitable microenvironment for EMT progression. FBXO32 is also amplified in metastatic cancers and its depletion in a NSG mouse xenograft model inhibits tumor growth and metastasis. In addition, FBXO32 is essential for neuronal EMT during brain development. Together, these findings establish that FBXO32 acts as an upstream regulator of EMT by governing the gene expression program underlying this process during development and disease.

ERK signalling modulates epigenome to drive epithelial to mesenchymal transition.

The series of events that allow the conversion from adherent epithelial cells into migratory cells is collectively known as epithelial-mesenchymal transition (EMT). EMT is employed during embryonic development such as for gastrulation and neural crest migration and is misused in diseases, such as cancer metastasis. ERK signalling is known to be essential for EMT, however its influence on the epigenetic and transcriptional programme underlying EMT is poorly understood. Here, using a comprehensive genome-wide analysis of H3K27ac mark and gene expression in mammary epithelial cells undergoing EMT, we found that ERK signalling is essential for the epigenetic reprogramming underlying hallmark gene expression and phenotypic changes of EMT. We show that the chemical inhibition of Erk signalling during EMT prevents the loss and gain of the H3K27ac mark at regulatory regions of epithelial and mesenchymal genes, respectively, and results in a transcriptome and epigenome closer to those of epithelial cells. Further computational analyses identified a distinct set of transcription factor motifs enriched at distal regulatory regions that are epigenetically remodelled by ERK signalling. Altogether, our findings reveal an ERK-dependent epigenetic remodelling of regulatory elements that results in a gene expression programme essential for driving EMT.

TOX3 regulates neural progenitor identity.

The human genomic locus for the transcription factor TOX3 has been implicated in susceptibility to restless legs syndrome and breast cancer in genome-wide association studies, but the physiological role of TOX3 remains largely unknown. We found Tox3 to be predominantly expressed in the developing mouse brain with a peak at embryonic day E14 where it co-localizes with the neural stem and progenitor markers Nestin and Sox2 in radial glia of the ventricular zone and intermediate progenitors of the subventricular zone. Tox3 is also expressed in neural progenitor cells obtained from the ganglionic eminence of E15 mice that express Nestin, and it specifically binds the Nestin promoter in chromatin immunoprecipitation assays. In line with this, over-expression of Tox3 increased Nestin promoter activity, which was cooperatively enhanced by treatment with the stem cell self-renewal promoting Notch ligand Jagged and repressed by pharmacological inhibition of Notch signaling. Knockdown of Tox3 in the subventricular zone of E12.5 mouse embryos by in utero electroporation of Tox3 shRNA revealed a reduced Nestin expression and decreased proliferation at E14 and a reduced migration to the cortical plate in E16 embryos in electroporated cells. Together, these results argue for a role of Tox3 in the development of the nervous system.

Dynamics of chromatin accessibility and epigenetic state in response to UV damage.

Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity.

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity.

JNK-dependent gene regulatory circuitry governs mesenchymal fate.

The epithelial to mesenchymal transition (EMT) is a biological process in which cells lose cell-cell contacts and become motile. EMT is used during development, for example, in triggering neural crest migration, and in cancer metastasis. Despite progress, the dynamics of JNK signaling, its role in genomewide transcriptional reprogramming, and involved downstream effectors during EMT remain largely unknown. Here, we show that JNK is not required for initiation, but progression of phenotypic changes associated with EMT. Such dependency resulted from JNK-driven transcriptional reprogramming of critical EMT genes and involved changes in their chromatin state. Furthermore, we identified eight novel JNK-induced transcription factors that were required for proper EMT. Three of these factors were also highly expressed in invasive cancer cells where they function in gene regulation to maintain mesenchymal identity. These factors were also induced during neuronal development and function in neuronal migration in vivo. These comprehensive findings uncovered a kinetically distinct role for the JNK pathway in defining the transcriptome that underlies mesenchymal identity and revealed novel transcription factors that mediate these responses during development and disease.

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5' donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy.

Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance.

The asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) acts as a repressive mark that antagonizes trimethylation of H3 lysine 4. Here we report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7 and present in euchromatic regions. Profiling of H3-tail interactors by SILAC MS revealed that H3R2me2s excludes binding of RBBP7, a central component of co-repressor complexes Sin3a, NURD and PRC2. Conversely H3R2me2s enhances binding of WDR5, a common component of the coactivator complexes MLL, SET1A, SET1B, NLS1 and ATAC. The interaction of histone H3 with WDR5 distinguishes H3R2me2s from H3R2me2a, which impedes the recruitment of WDR5 to chromatin. The crystallographic structure of WDR5 and the H3R2me2s peptide elucidates the molecular determinants of this high affinity interaction. Our findings identify H3R2me2s as a previously unknown mark that keeps genes poised in euchromatin for transcriptional activation upon cell-cycle withdrawal and differentiation in human cells.