Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a

Overexpression of Arabidopsis Dehydration Response Element Binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. While the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be colocalized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding when compared to non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a overexpressing lines, suggesting antagonism between heat and dehydration stress pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and rapid response towards stress tolerance as well as attenuation of the signal to prevent deleterious effects of the runoff response.

RNA polymerase II pausing in development: orchestrating transcription

The coordinated regulation of transcriptional networks underpins cellular identity and developmental progression. RNA polymerase II promoter-proximal pausing (Pol II pausing) is a prevalent mechanism by which cells can control and synchronize transcription. Pol II pausing regulates the productive elongation step of transcription at key genes downstream of a variety of signalling pathways, such as FGF and Nodal. Recent advances in our understanding of the Pol II pausing machinery and its role in transcription call for an assessment of these findings within the context of development. In this review, we discuss our current understanding of the molecular basis of Pol II pausing and its function during organismal development. By critically assessing the tools used to study this process we conclude that combining recently developed genomics approaches with refined perturbation systems has the potential to expand our understanding of Pol II pausing mechanistically and functionally in the context of development and beyond.

Rapid Cis–Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4–NOT Component in Drosophila

Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4–NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. Here, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, we found a dynamic pattern in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which we term Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, we found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). Our results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. Our results thus uncover dynamic genome-wide evolutionary processes associated with new genes.

Transcriptional Networks Identify BRPF1 as a Potential Drug Target Based on Inflammatory Signature in Primary Lower-Grade Gliomas

Gliomas are the most common tumors of the central nervous system and are classified into grades I-IV based on their histological characteristics. Lower-grade gliomas (LGG) can be divided into grade II diffuse low-grade gliomas and grade III moderate gliomas and have a relatively good prognosis. However, LGG often develops into high-grade glioma within a few years. This study aimed to construct and identify the prognostic value of an inflammatory signature and discover potential drug targets for primary LGG. We first screened differentially expressed genes in primary LGG (TCGA) compared with normal brain tissue (GTEx) that overlapped with inflammation-related genes from MSigDB. After survival analysis, nine genes were selected to construct an inflammatory signature. LGG patients with a high inflammatory signature score had a poor prognosis, and the inflammatory signature was a strong independent prognostic factor in both the training cohort (TCGA) and validation cohort (CGGA). Compared with the low-inflammatory signature group, differentially expressed genes in the high-inflammatory signature group were mainly enriched in immune-related signaling pathways, which is consistent with the distribution of immune cells in the high- and low-inflammatory signature groups. Integrating driver genes, upregulated genes and drug targets data, bromodomain and PHD finger-containing protein 1 (BRPF1) was selected as a potential drug target. Inhibition of BRPF1 function or knockdown of BRPF1 expression attenuated glioma cell proliferation and colony formation.

Targeting Oncogenic Transcriptional Networks in Neuroblastoma: From N-Myc to Epigenetic Drugs

Neuroblastoma (NB) is one of the most frequently occurring neurogenic extracranial solid cancers in childhood and infancy. Over the years, many pieces of evidence suggested that NB development is controlled by gene expression dysregulation. These unleashed programs that outline NB cancer cells make them highly dependent on specific tuning of gene expression, which can act co-operatively to define the differentiation state, cell identity, and specialized functions. The peculiar regulation is mainly caused by genetic and epigenetic alterations, resulting in the dependency on a small set of key master transcriptional regulators as the convergence point of multiple signalling pathways. In this review, we provide a comprehensive blueprint of transcriptional regulation bearing NB initiation and progression, unveiling the complexity of novel oncogenic and tumour suppressive regulatory networks of this pathology. Furthermore, we underline the significance of multi-target therapies against these hallmarks, showing how novel approaches, together with chemotherapy, surgery, or radiotherapy, can have substantial antineoplastic effects, disrupting a wide variety of tumorigenic pathways through combinations of different treatments.

Identification of ASCL1 as a determinant for human iPSC-derived dopaminergic neurons

During cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.

Mouse strain-specific polymorphic provirus functions as cis-regulatory element leading to epigenomic and transcriptomic variations

Polymorphic integrations of endogenous retroviruses (ERVs) have been previously detected in mouse and human genomes. While most are inert, a subset can influence the activity of the host genes. However, the molecular mechanism underlying how such elements affect the epigenome and transcriptome and their roles in driving intra-specific variation remain unclear. Here, by utilizing wildtype murine embryonic stem cells (mESCs) derived from distinct genetic backgrounds, we discover a polymorphic MMERGLN (GLN) element capable of regulating H3K27ac enrichment and transcription of neighboring loci. We demonstrate that this polymorphic element can enhance the neighboring Klhdc4 gene expression in cis, which alters the activity of downstream stress response genes. These results suggest that the polymorphic ERV-derived cis-regulatory element contributes to differential phenotypes from stimuli between mouse strains. Moreover, we identify thousands of potential polymorphic ERVs in mESCs, a subset of which show an association between proviral activity and nearby chromatin states and transcription. Overall, our findings elucidate the mechanism of how polymorphic ERVs can shape the epigenome and transcriptional networks that give rise to phenotypic divergence between individuals.

NFATC2 regulates Targets of MYC Signaling in MLL-AF9 AML

Background: Acute myeloid leukemia (AML) arises due to an accumulation of genetic lesions within myeloid progenitors and oncogenic transformation is often characterised by disordered transcription. Recently the histone lysine demethylase KDM4A was shown to be essential for AML blast survival and self-renewal. shRNA knockdown (KD) of KDM4A led to downregulated expression of the transcription factor NFATC2 an MLL-AF9 AML model, suggesting that it is a key target of KDM4A oncogenic function. The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors control cell cycle genes and self-renewal pathways in hematopoietic tissues and are well-defined as oncogenic regulators in various malignancies. NFATs have recently been attributed roles in the development of FLT3 ITD AML and resistance to tyrosine kinase inhibitors (TKIs) in myeloid leukemias but there is little evidence detailing the role(s) of NFATC2 specifically in AML. We hypothesized that NFATc2 activity is essential for the survival of AML cells and the oncogenic transcriptional networks within these. Aims: To determine if AML cells are dependent on NFATC2 for survival and to elucidate the transcriptional and binding targets of NFATc2 in AML. Methods: NFATC2 was depleted using shRNA KD in numerous cell line models of AML and putative transcriptional targets were elucidated using RNA-seq following KD. Binding targets of NFATc2 were determined using ChIP-seq. Transcriptomic targets of NFATc2 were validated using the Fluidigm Biomark multiplex PCR system and real time quantitative PCR. Results: KD of NFATC2 significantly impaired the colony forming capacity and expansion in liquid cultures of AML cell lines from diverse (cyto)genetic backgrounds. MLL-AF9/TP53 mut THP-1 cells showed reduced entry to the S-phase of the cell cycle and downregulation of cyclin D1 following NFATC2 depletion, suggesting that NFATC2 is critical for cell cycle progression in these cells. Overexpression of human NFATC2 in THP-1 led to an increased rate of cell growth. RNA-seq analysis of THP-1 cells with NFATC2 KD revealed >20 genes with deregulated expression (FDR<0.1), which have been validated using PCR methods. Overexpression of human NFATC2 resulted in significant deregulation of 9 of these genes (FDR<0.1), defining a subset of genes which may regulate the observed phenotype. Additionally, these top genes were not all differentially regulated in other MLL-AF9 AML cell lines MOLM-13 and NOMO-1 following NFATC2 KD. Finally, in THP-1, gene set enrichment analysis (GSEA) of sequencing results revealed that targets of MYC and calmodulin kinase STK33 were enriched within the genes perturbed by NFATC2 depletion. Targets of MYC signaling were validated by PCR in THP-1 but were not found to be deregulated in MOLM-13 following NFATC2 KD. ChIP-seq analysis of NFATc2 binding in THP-1 cells showed that >30% of NFATc2 targets were at promoter regions within 5kb of the transcription start site. Motif analysis of precipitated DNA fragments discovered two novel motifs which were enriched at NFATc2 binding sites (p<0.0001). Discussion: NFATC2 was found to be essential for expansion of AML cells in various cell line models. In the MLL-AF9 driven THP-1 model a number of putative transcriptional and genomic targets were defined, which include novel targets not previously described in AML pathogenesis and targets of MYC, an established oncogenic protein in AML. The differing expression profiles observed across AML cell lines of diverse (cyto)genetic backgrounds with NFATC2 KD suggest that the regulatory targets of NFATc2 vary depending on the cellular signaling landscape. Together with the finding that NFATC2 is indispensable for AML cell survival this study has elucidated novel roles(s) for NFATC2 in AML oncogenesis. Disclosures Massett: Kymab Ltd: Current Employment. Huang: Janssen Pharmaceutical Companies of Johnson & Johnson (China): Current Employment.

High Mobility Group A1 (HMGA1) Epigenetic Regulators Induce ETV5 Networks in Relapsed B-Cell Leukemia and Provide Novel Therapeutic Targets

Introduction: Despite advances in therapy for B-cell acute lymphoblastic leukemia (B-ALL), relapsed disease remains the leading cause of death in children with cancer. The gene encoding the High Mobility Group A1 (HMGA1) chromatin regulator is highly expressed in stem cells and diverse malignancies where high levels portend poor outcomes. We discovered that transgenic mice misexpressing Hmga1 in lymphoid cells develop leukemic transformation by amplifying transcriptional networks involved in stem cell function, proliferation, and inflammation (Hillion et al, Cancer Res 2008, Schuldenfrei et al, BMC Genomics 2011, Xian et al, Nature Commun 2017). In pediatric B-ALL (pB-ALL), HMGA1 is overexpressed with highest levels in blasts from early relapse (Roy et al, Leuk Lymphoma 2013). Together, these findings suggest that HMGA1 is required for leukemogenesis and drives relapse through epigenetic reprogramming. We therefore sought to: 1) test the hypothesis that HMGA1 is required for leukemogenesis and relapse in pB-ALL, and, 2) elucidate targetable mechanisms mediated by HMGA1. Methods: To elucidate the function of HMGA1 and downstream targets, we employed CRISPR/Cas9 gene inactivation and lentiviral-mediated gene silencing via delivery of short hairpin RNA (shRNA) targeting 2 sequences per gene in cell lines from relapsed pB-ALL, including REH, which harbor the TEL-AML1 fusion, and 697, which harbor the E2A-PBX1 fusion. We assessed leukemia phenotypes in vitro and leukemic engraftment in vivo. To dissect molecular mechanisms, we performed RNA sequencing (RNAseq) and applied in silico pathway analysis. To validate these pathways in human pB-ALL, we assessed gene expression and clinical outcomes in independent cohorts. The Broad Institute Connectivity Map (CMAP) was applied to identify drugs to target HMGA1 networks. Results: HMGA1 is overexpressed in pB-ALL in independent cohorts with highest levels at relapse. Decreasing HMGA1 expression via CRISPR/Cas9 inactivation or shRNA-mediated gene silencing in relapsed pB-ALL cell lines (REH, 697) disrupts proliferation, decreases the frequency of cells in S phase concurrent with increases in G0/G1, enhances apoptosis, and impairs clonogenicity. To assess HMGA1 function in vivo, we compared leukemogenesis following tail vein injection of pB-ALL cell lines with or without HMGA1 depletion in immunodeficient mice (NOD/SCID/IL2 receptor gamma null). Survival was prolonged in mice injected with either pB-ALL cell line (REH, 697) after HMGA1 depletion. Further, leukemic cells that ultimately engraft show increased HMGA1 expression relative to the pool of injected cells with HMGA1 silencing, suggesting that escape from HMGA1 silencing was required for engraftment. RNAseq revealed transcriptional networks governed by HMGA1 that regulate proliferation (G2M checkpoint, E2F), RAS/ERK signaling, hematopoietic stem cells, and ETV5 (ETS variant 5 transcription factor) targets. Given its association with aggressive ALL harboring the BCR-ABL fusion, we focused on the ETV5 gene. CRISPR/Cas9 inactivation or gene silencing of ETV5 in relapsed pB-ALL cell lines (REH, 697) decreases proliferation and clonogenicity in vitro, while delaying leukemogenesis in vivo. Further, restoring ETV5 expression in pB-ALL cell lines with HMGA1 silencing partially rescues anti-leukemogenic effects of HMGA1 depletion. Mechanistically, HMGA1 binds to AT-rich regions within the ETV5 promoter (-0.7 kb and -0.2 kb) and recruits active histone marks (H3K27Ac, H3K4me3, H3K4me1) to induce ETV5. Epigenetic drugs predicted to target HMGA1-ETV5 networks synergize with HMGA1 silencing in cytotoxicity assays with pB-ALL cell lines. Most importantly, HMGA1 and ETV5 are co-expressed and up-regulated in primary blasts from children with pB-ALL with highest levels at relapse, thus underscoring the significance of this pathway in relapsed pediatric B-ALL. Conclusions: We discovered a previously unknown epigenetic program whereby HMGA1 up-regulates ETV5 networks by binding to chromatin and recruiting active histone marks to the ETV5 promoter. Both HMGA1 and ETV5 are up-regulated at relapse. Finally, the HMGA1-ETV5 axis can be targeted by epigenetic drugs (HDAC inhibitors) that synergize with HMGA1 depletion. Our findings reveal the HMGA1-ETV5 axis as a key molecular switch in relapsed pB-ALL and rational therapeutic target to treat or prevent relapse. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Targeting SerpinE1 reverses cellular features of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of perinuclear heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. However, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. We have performed a longitudinal genome-wide analysis of gene expression in primary HGPS fibroblasts from patients at two sequential stages of disease that revealed a progressive activation of Rho signaling and SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1). siRNA-mediated downregulation or pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by reduced levels of Progerin and correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector of HGPS pathogenesis and target for therapeutic interventions.