Recruitment Of Chromatin Modifiers Research Articles

The B-box protein CmBBX8 recruits chromatin modifiers CmFDM2/CmSWI3B to induce flowering in summer chrysanthemum.

The transition from vegetative to reproductive growth is essential for the flowering process of plants. In summer chrysanthemum, CmBBX8 exploits prominence function in floral transition by activating the expression of CmFTL1. However, how CmBBX8 induces CmFTL1 during the photoperiod inductive cycles remains unknown. Here, we show that CmBBX8 interacts with the SGS3-like protein CmFDM2, and the CmFDM2 overexpression strains presented early flowering, while knockdown strains delayed flowering. Additionally, CmFDM2 could bind to the CmFTL1 promoter and activate the expression of CmFTL1, and associate with chromatin remodeling factor CmSWI3B, and CmBBX8 induces flowering dependent on CmFDM2 and CmSWI3B. CmFDM2 also partially depends on CmSWI3B. The CmSWI3B knockdown strains exhibited a significant late flowering phenotype. Interestingly, CmBBX8 also interacts with CmSWI3B. Moreover, the level of H3K27me3 at the CmFTL1 locus was reduced when CmBBX8 and CmFDM2/CmSWI3B occupied the locus to promote chrysanthemum flowering during the photoperiod inductive cycles, which was accompanied by the increasing level of CmFTL1 transcripts. Thus, our work provides novel insights into the gradually increasing level of CmFTL1 for the floral transition through CmBBX8 recruiting chromatin modifiers CmFDM2/CmSWI3B.

Noncoding RNAs: Emerging regulators of behavioral complexity.

The mammalian genome encodes thousands of non-coding RNAs (ncRNAs), ranging in size from about 20 nucleotides (microRNAs or miRNAs) to kilobases (long non-coding RNAs or lncRNAs). ncRNAs contribute to a layer of gene regulation that could explain the evolution of massive phenotypic complexity even as the number of protein-coding genes remains unaltered. We propose that low conservation, poor expression, and highly restricted spatiotemporal expression patterns-conventionally considered ncRNAs may affect behavior through direct, rapid, and often sustained regulation of gene expression at the transcriptional, post-transcriptional, or translational levels. Besides these direct roles, their effect during neurodevelopment may manifest as behavioral changes later in the organism's life, especially when exposed to environmental cues like stress and seasonal changes. The lncRNAs affect behavior through diverse mechanisms like sponging of miRNAs, recruitment of chromatin modifiers, and regulation of alternative splicing. We highlight the need for synthesis between rigorously designed behavioral paradigms in model organisms and the wide diversity of behaviors documented by ethologists through field studies on organisms exquisitely adapted to their environmental niche. Comparative genomics and the latest advancements in transcriptomics provide an unprecedented scope for merging field and lab studies on model and non-model organisms to shed light on the role of ncRNAs in driving the behavioral responses of individuals and groups. We touch upon the technical challenges and contentious issues that must be resolved to fully understand the role of ncRNAs in regulating complex behavioral traits. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Expression of collagen-related piRNA is dysregulated in cultured dermal fibroblasts derived from patients with scleroderma.

PIWI-interacting RNA (piRNA) is a class of recently discovered small non-coding RNAs. piRNAs derive from an initial transcript encompassing a piRNA cluster via a unique biosynthesis process, interact with PIWI proteins, bind to specific targets, and recruit chromatin modifiers to enable transcriptional repression. Abnormal expression of PIWI proteins and piRNAs has been reported in some human cancers, with participation of some PIWI/piRNAs complexes in tumorigenesis and association with cancer prognosis. Their expression in patients with systemic sclerosis (SSc) has not been widely elucidated. PIWI/piRNAs and their role in the pathogenesis of collagen accumulation in SSc was therefore investigated; no difference was found in the PIWIL1-4 levels between normal and cultured SSc dermal fibroblasts. Among piRNAs predicted to target SSc-related molecules, we first found significant piR-32364 up-regulation in SSc dermal fibroblasts, likely due to intrinsic TGF-β signaling. Forced piR-32364 overexpression in normal fibroblasts significantly reduced COL1A1 expression both at mRNA and protein levels, but not COL1A2. Thus, piR-32364 overexpression in SSc fibroblasts may be the negative feedback against collagen up-regulation, which could suggest the potential of piRNAs as a therapeutic target.

Interaction of hnRNPB1 with Helix-12 of hHOTAIR Reveals the Distinctive Mode of RNA Recognition That Enables the Structural Rearrangement by LCD.

The lncRNA human Hox transcript antisense intergenic RNA (hHOTAIR) regulates gene expression by recruiting chromatin modifiers. The prevailing model suggests that hHOTAIR recruits hnRNPB1 to facilitate intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target gene transcripts. This B1-mediated RNA-RNA interaction modulates the structure of hHOTAIR, attenuates its inhibitory effect on polycomb repression complex 2, and enhances its methyl transferase activity. However, the molecular details by which the nuclear hnRNPB1 protein assembles on the lncRNA HOTAIR have not yet been described. Here, we investigate the molecular interactions between hnRNPB1 and Helix-12 (hHOTAIR). We show that the low-complexity domain segment (LCD) of hnRNPB1 interacts with a strong affinity for Helix-12. Our studies revealed that unbound Helix-12 folds into a specific base-pairing pattern and contains an internal loop that, as determined by thermal melting and NMR studies, exhibits hydrogen bonding between strands and forms the recognition site for the LCD segment. In addition, mutation studies show that the secondary structure of Helix-12 makes an important contribution by acting as a landing pad for hnRNPB1. The secondary structure of Helix-12 is involved in specific interactions with different domains of hnRNPB1. Finally, we show that the LCD unwinds Helix-12 locally, indicating its importance in the hHOTAIR restructuring mechanism.

Dynamic chromatin accessibility deploys heterotypic cis/trans-acting factors driving stomatal cell-fate commitment

Chromatin architecture and transcription factor (TF) binding underpin cell-fate specification during development, but their mutual regulatory relationships remain unclear. Here we report an atlas of dynamic chromatin landscapes during stomatal cell-lineage progression, in which sequential cell-state transitions are governed by lineage-specific bHLH TFs. Major reprogramming of chromatin accessibility occurs at the proliferation-to-differentiation transition. We discover novel co-cis regulatory elements (CREs) signifying the early precursor stage, BBR/BPC (GAGA) and bHLH (E-box) motifs, where master-regulatory bHLH TFs, SPEECHLESS and MUTE, consecutively bind to initiate and terminate the proliferative state, respectively. BPC TFs complex with MUTE to repress SPEECHLESS expression through a local deposition of repressive histone marks. We elucidate the mechanism by which cell-state-specific heterotypic TF complexes facilitate cell-fate commitment by recruiting chromatin modifiers via key co-CREs.

Sociosexual behavior requires both activating and repressive roles of Tfap2e/AP-2ε in vomeronasal sensory neurons.

Neuronal identity dictates the position in an epithelium, and the ability to detect, process, and transmit specific signals to specified targets. Transcription factors (TFs) determine cellular identity via direct modulation of genetic transcription and recruiting chromatin modifiers. However, our understanding of the mechanisms that define neuronal identity and their magnitude remain a critical barrier to elucidate the etiology of congenital and neurodegenerative disorders. The rodent vomeronasal organ provides a unique system to examine in detail the molecular mechanisms underlying the differentiation and maturation of chemosensory neurons. Here, we demonstrated that the identity of postmitotic/maturing vomeronasal sensory neurons (VSNs), and vomeronasal-dependent behaviors can be reprogrammed through the rescue of Tfap2e/AP-2ε expression in the Tfap2eNull mice, and partially reprogrammed by inducing ectopic Tfap2e expression in mature apical VSNs. We suggest that the TF Tfap2e can reprogram VSNs bypassing cellular plasticity restrictions, and that it directly controls the expression of batteries of vomeronasal genes.

Crosstalk Between Histone and m6A Modifications and Emerging Roles of m6A RNA Methylation.

RNA, like DNA and proteins, has been discovered to undergo dynamic and reversible chemical alterations, increasing the diversity and functional complexity of the molecule. N-6-methyladenosine (m6A) RNA methylation serves as a bridge between transcription and translation and is critical for many diseases’ progression. There is a complex interrelationship between m6A modifications and other epigenetic modifications. Their crosstalk significantly affects transcriptional outputs, translation, recruitment of chromatin modifiers, as well as the deployment of the m6A methyltransferase complex at target sites. This article outlines the potential function of m6A RNA methylation in epigenetics and summarizes its interactions with histone modifications.

LncRNA HOTAIR: A Potential Prognostic Factor and Therapeutic Target in Human Cancers.

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of gene expression and physiological processes. LncRNAs are a class of ncRNAs of 200 nucleotides in length. HOX transcript antisense RNA (HOTAIR), a trans-acting lncRNA with regulatory function on transcription, can repress gene expression by recruiting chromatin modifiers. HOTAIR is an oncogenic lncRNA, and numerous studies have determined that HOTAIR is highly upregulated in a wide variety of human cancers. In this review, we briefly summarize the impact of lncRNA HOTAIR expression and functions on different human solid tumors, and emphasize the potential of HOTAIR on tumor prognosis and therapy. Here, we review the recent studies that highlight the prognostic potential of HOTAIR in drug resistance and survival, and the progress of therapies developed to target HOTAIR to date. Furthermore, targeting HOTAIR results in the suppression of HOTAIR expression or function. Thus, HOTAIR knockdown exhibits great therapeutic potential in various cancers, indicating that targeting lncRNA HOTAIR may serve as a promising strategy for cancer therapy. We also propose that preclinical studies involving HOTAIR are required to provide a better understanding of the exact molecular mechanisms underlying the dysregulation of its expression and function in different human cancers and to explore effective methods of targeting HOTAIR and engineering efficient and targeted drug delivery methods in vivo.

Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading.

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.

Insight Into Chromatin-Enriched RNA: A Key Chromatin Regulator in Tumors.

Chromatin-enriched RNAs (cheRNAs) constitute a special class of long noncoding RNAs (lncRNAs) that are enriched around chromatin and function to activate neighboring or distal gene transcription. Recent studies have shown that cheRNAs affect chromatin structure and gene expression by recruiting chromatin modifiers or acting as bridges between distal enhancers and promoters. The abnormal transcription of cheRNAs plays an important role in the occurrence of many diseases, particularly tumors. The critical effect of cancer stem cells (CSCs) on the formation and development of tumors is well known, but the function of cheRNAs in tumorigenesis, especially in CSC proliferation and stemness maintenance, is not yet fully understood. This review focuses on the mechanisms of cheRNAs in epigenetic regulation and chromatin conformation and discusses the way cheRNAs function in CSCs to deepen the understanding of tumorigenesis and provide novel insight to advance tumor-targeting therapy.

Multifaceted Actions of GFI1 and GFI1B in Hematopoietic Stem Cell Self-Renewal and Lineage Commitment.

Growth factor independence 1 (GFI1) and the closely related protein GFI1B are small nuclear proteins that act as DNA binding transcriptional repressors. Both recognize the same consensus DNA binding motif via their C-terminal zinc finger domains and regulate the expression of their target genes by recruiting chromatin modifiers such as histone deacetylases (HDACs) and demethylases (LSD1) by using an N-terminal SNAG domain that comprises only 20 amino acids. The only region that is different between both proteins is the region that separates the zinc finger domains and the SNAG domain. Both proteins are co-expressed in hematopoietic stem cells (HSCs) and, to some extent, in multipotent progenitors (MPPs), but expression is specified as soon as early progenitors and show signs of lineage bias. While expression of GFI1 is maintained in lymphoid primed multipotent progenitors (LMPPs) that have the potential to differentiate into both myeloid and lymphoid cells, GFI1B expression is no longer detectable in these cells. By contrast, GFI1 expression is lost in megakaryocyte precursors (MKPs) and in megakaryocyte-erythrocyte progenitors (MEPs), which maintain a high level of GFI1B expression. Consequently, GFI1 drives myeloid and lymphoid differentiation and GFI1B drives the development of megakaryocytes, platelets, and erythrocytes. How such complementary cell type- and lineage-specific functions of GFI1 and GFI1B are maintained is still an unresolved question in particular since they share an almost identical structure and very similar biochemical modes of actions. The cell type-specific accessibility of GFI1/1B binding sites may explain the fact that very similar transcription factors can be responsible for very different transcriptional programming. An additional explanation comes from recent data showing that both proteins may have additional non-transcriptional functions. GFI1 interacts with a number of proteins involved in DNA repair and lack of GFI1 renders HSCs highly susceptible to DNA damage-induced death and restricts their proliferation. In contrast, GFI1B binds to proteins of the beta-catenin/Wnt signaling pathway and lack of GFI1B leads to an expansion of HSCs and MKPs, illustrating the different impact that GFI1 or GFI1B has on HSCs. In addition, GFI1 and GFI1B are required for endothelial cells to become the first blood cells during early murine development and are among those transcription factors needed to convert adult endothelial cells or fibroblasts into HSCs. This role of GFI1 and GFI1B bears high significance for the ongoing effort to generate hematopoietic stem and progenitor cells de novo for the autologous treatment of blood disorders such as leukemia and lymphoma.

Long Non-Coding RNA Expression in Waldenstrom Macroglobulinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Long Non-Coding RNA Expression in Waldenstrom Macroglobulinemia and IgM Monoclonal Gammopathy of Undetermined Significance

The CAF-1 complex couples Hippo pathway target gene expression and DNA replication

The Hippo signaling pathway regulates tissue growth and organ development in many animals, including humans. Pathway activity leads to inactivation of Yorkie (Yki), a transcriptional coactivator that drives expression of growth-promoting genes. In addition, Yki has been shown to recruit chromatin modifiers that enhance chromatin accessibility and thereby enhance Yki function. Here, we asked whether changes in chromatin accessibility that occur during DNA replication could also affect Yki function. We found that depletion of the chromatin assembly complex-1 (CAF-1) complex, a histone chaperone that is required for nucleosome assembly after DNA replication, in the wing imaginal epithelium leads to increased Hippo pathway target gene expression but does not affect expression of other genes. Yki shows greater association with target sites when CAF-1 is depleted and misregulation of target gene expression is Yki-dependent, suggesting that nucleosome assembly competes with Yki for pathway targets post-DNA replication. Consistent with this idea, increased target gene expression is DNA replication dependent and newly replicated chromatin at target sites shows marked nucleosome depletion when CAF-1 function is reduced. These observations suggest a connection between cell cycle progression and Hippo pathway target expression, providing insights into functions of the Hippo pathway in normal and abnormal tissue growth.

S110 SCREENING OF CLUSTERED REGULATORY ELEMENTS REVEALS NOVEL LEUKEMIA DEPENDENCIES

Background:In the recent years, massively parallel sequencing approaches allowed the identification of hundreds of mutated genes in Leukemia. Although these data gave unprecedented amount of information about mechanisms of leukemia cell maintenance and/or progression, the functional characterization of genes that are key player in regulating cancer development remain laborious. Analysis at the single gene level often fails to identify gene or pathway collaborations leading to transformation. Studies aimed at depicting new oncogene cooperation would involve the generation of challenging mouse models or the deployment of tedious screening pipelines, which would be inadequate to depict new oncogene circuitry in cancer. Genome wide mapping of epigenetic modifications on histone tails or binding of factors such as MED1 and BRD4 allowed identification of clusters of regulatory elements, also termed as Super Enhancers. Functional annotation of these regions revealed their high relevance during normal hematopoiesis and Leukemogenesis.Aims:We hypothesized that these regulatory regions could regulate simultaneously expression of genes cooperating to promote Leukemia development. We therefore aimed to functionally screen these Super Enhancers (SE) and define what genes they directly regulates and how they collaborate in leukemia pathogenesis.Methods:We developed a novel genome‐wide CRISPRi‐based screening approach to directly target these regulatory regions. CRISPRi technology relies on the use of deactivated Cas9 that can’t cut the DNA and that is fused to the repressive KRAB domain. Therefore, properly targeted dCas9‐KRAB by single guide RNAs will recruit chromatin modifying factors and trigger generation of heterochromatin thus inhibiting enhancer function. We performed this screen using acute megakaryoblastic leukemia model driven by the CBFA2T3‐GLIS2 fusion, the most frequent fusion oncogene in this disease that we recently identified as being associated with Super Enhancers (Thirant et al, Cancer Cell 2017). To inhibit SE activity we integrated ChIP‐seq data of H3K27ac and ATAC‐seq data to define open chromatin regions located in Super Enhancers. We designed a library of 7995 single guide RNAs targeting 450 SE regions found active in CBFA2T3‐GLIS2 bearing cell line M07e and primary AMKL patient samples.Results:This screening methodology allowed us to nominate SE regions, which are functionally linked to leukemia progression. In particular, we pinpointed a novel SE region, induced by CBFA2T3‐GLIS2 fusion, regulating the expression of both tyrosine kinases associated receptors KIT and PDGFRA. We were able to show that this SE region is normally not active in megakaryocytic development and aberrantly induced by CBFA2T3‐GLIS2 expression. RNA‐seq experiments and 4C‐seq experiments (chromatin conformation capture) showed that this SE is directly regulating KIT and PDGFRA expression. Whereas single inhibition of these genes using shRNA or small molecule inhibitors affects modestly leukemic cell growth, concomitant inhibition of these two receptors synergizes to impair AMKL cell lines and primary patient cells growth and survival.In vivo targeting of this Super Enhancer activity in patient‐derived xenograft models using CRISPRi showed significant reduction of tumor burden and increased overall survival.Summary/Conclusion:Our results demonstrate that genome‐wide screening of regulatory DNA elements can identify co‐regulated genes collaborating to promote leukemia progression and could open new avenues for the design of combination therapies.

Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins.

Rearrangement of the 1q12 pericentromeric heterochromatin and subsequent amplification of the 1q arm is commonly associated with cancer development and progression and may result from epigenetic deregulation. In many premalignant and malignant cells, loss of 1q12 satellite DNA methylation causes the deposition of polycomb factors and formation of large polycomb aggregates referred to as polycomb bodies. Here, we show that SSX proteins can destabilize 1q12 pericentromeric heterochromatin in melanoma cells when it is present in the context of polycomb bodies. We found that SSX proteins deplete polycomb bodies and promote the unfolding and derepression of 1q12 heterochromatin during replication. This further leads to segregation abnormalities during anaphase and generation of micronuclei. The structural rearrangement of 1q12 pericentromeric heterochromatin triggered by SSX2 is associated with loss of polycomb factors, but is not mediated by diminished polycomb repression. Instead, our studies suggest a direct effect of SSX proteins facilitated though a DNA/chromatin binding, zinc finger-like domain and a KRAB-like domain that may recruit chromatin modifiers or activate satellite transcription. Our results demonstrate a novel mechanism for generation of 1q12-associated genomic instability in cancer cells.

Replication-Independent Histone Turnover Underlines the Epigenetic Homeostasis in Adult Heart.

Replication-independent histone turnover has been linked to cis-regulatory chromatin domains in cultured cell lines, but its molecular underpinnings and functional relevance in adult mammalian tissues remain yet to be defined. We investigated regulatory functions of replication-independent histone turnover in chromatin states of postmitotic cardiomyocytes from adult mouse heart. We used H2B-GFP (histone 2B-green fluorescent protein) fusion protein pulse-and-chase approaches to measure histone turnover rate in mouse cardiomyocytes. Surprisingly, we found that the short histone half-life (≈2 weeks) contrasted dramatically with the long lifetime of cardiomyocytes, and rapid histone turnover regions corresponded to cis-regulatory domains of heart genes. Interestingly, recruitment of chromatin modifiers, including Polycomb EED (embryonic ectoderm development), was positively correlated with histone turnover rate at enhancers. Mechanistically, through directly interacting with and engaging the BAF (BRG1 [Brahma-related gene-1]-associated factor) complex for nucleosome exchange for stereotyped histone modifications from the free histone pool, EED augmented histone turnover to restrain enhancer overactivation. We propose a model in which replication-independent histone turnover reinforces robustness of local chromatin states for adult tissue homeostasis.

A Dissection of Oligomerization by the TRIM28 Tripartite Motif and the Interaction with Members of the Krab-ZFP Family

TRIM28 (also known as KAP1 or TIF1β) is the universal co-repressor of the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs), the largest family of transcription factors in mammals. During early embryogenesis, TRIM28 mediates the transcriptional silencing of many endogenous retroviral elements and genomic imprinted sites. Silencing is initiated by the recruitment of TRIM28 to a target locus by members of the Krab-ZFP. Subsequently, TRIM28 functions as a scaffold protein to recruit chromatin modifying effectors featuring SETDB1, HP1 and the NuRD complex. Although many protein partners involved in silencing have been identified, the molecular basis of the protein interactions that mediate silencing remains largely unclear. In the present study, we identified the first Bbox domain (T28_B1 135–203) as a molecular interface responsible for the formation of higher-order oligomers of TRIM28. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt the formation of oligomers but have no observed effect on transcriptional silencing defining a single TRIM28 dimer as the functional unit for silencing. Using assembly-deficient mutants, we employed small-angle X-ray scattering and biophysical techniques to characterize binding to member of the Krab-ZFP family. This allows us to narrow and define the binding interface to the center of the coiled-coil region (residues 294–321) of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins.

Crispri-Based Screening of Clustered Regulatory Elements Reveals Novel Leukemia Dependencies

In the recent years, massively parallel sequencing approaches allowed the identification of hundreds of mutated genes in Leukemia. Although these data gave unprecedented amount of information about mechanisms of leukemia cell maintenance and/or progression, the functional characterization of genes that are key player in regulating cancer development remain laborious. Analysis at the single gene level often fails to identify gene or pathway collaborations leading to transformation. Studies aimed at depicting new oncogene cooperation would involve the generation of challenging mouse models or the deployment of tedious screening pipelines, which would be inadequate to depict new oncogene circuitry in cancer. Genome wide mapping of epigenetic modifications on histone tails or binding of factors such as MED1 and BRD4 allowed identification of clusters of regulatory elements, also termed as Super Enhancers. Functional annotation of these regions revealed their high relevance during normal hematopoiesis and Leukemogenesis. We hypothesized that these regulatory regions could regulate simultaneously expression of genes cooperating to promote Leukemia development.We thus developed a novel genome-wide CRISPRi-based screening approach to directly target these regulatory regions. CRISPRi technology relies on the use of deactivated Cas9 that can't cut the DNA and that is fused to the repressive KRAB domain (dCas9-KRAB). Therefore, properly targeted dCas9-KRAB by single guide RNAs will recruit chromatin modifying factors and trigger generation of heterochromatin thus inhibiting enhancer function. We performed this screen using acute megakaryoblastic leukemia model driven by the CBFA2T3-GLIS2 fusion, the most frequent fusion oncogene in this disease that we recently identified as being associated with Super Enhancers (Thirant et al, Cancer Cell 2017). To inhibit Super Enhancer activity we integrated ChIP-seq data of H3K27ac and ATAC-seq data to define open chromatin regions located in Super Enhancers. We designed a library of 7995 single guide RNAs targeting 450 Super Enhancer regions found active in CBFA2T3-GLIS2 bearing cell line M07e and primary AMKL patient samples.This screening methodology allowed us to nominate Super Enhancer regions, which are functionally linked to leukemia progression. In particular, we pinpointed a novel Super Enhancer region, induced by CBFA2T3-GLIS2 fusion, regulating the expression of both tyrosine kinases associated receptors KIT and PDGFRA. We were able to show that this Super Enhancer region is normally not active in normal megakaryocytic development and aberrantly induced by CBFA2T3-GLIS2 expression. RNA-sequencing experiments and 4C-seq experiments (chromatin conformation capture) showed that this Super Enhancer is directly regulating KIT and PDGFRA expression. Whereas single inhibition of these genes using shRNA or small molecule inhibitors affects modestly leukemic cell growth, concomitant inhibition of these two receptors synergizes to impair AMKL cell lines and primary patient cells growth and survival.In vivo targeting of this Super Enhancer activity in patient-derived xenograft models using CRISPRi showed significant reduction of tumor burden and increased overall survival.Our results demonstrate that genome-wide screening of regulatory DNA elements can identify co-regulated genes collaborating to promote leukemia progression and could open new avenues for the design of combination therapies.Reference:Thirant C, Ignacimouttou C, Lopez CK, Diop M, Le Mouël L, Thiollier C, Siret A, Dessen P, Aid Z, Rivière J, Rameau P, Lefebvre C, Khaled M, Leverger G, Ballerini P, Petit A, Raslova H, Carmichael CL, Kile BT, Soler E, Crispino JD, Wichmann C, Pflumio F, Schwaller J, Vainchenker W, Lobry C, Droin N, Bernard OA, Malinge S, Mercher T (2017). ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia. Cancer Cell. 31(3):452-465. DisclosuresNo relevant conflicts of interest to declare.

RNA Immunoprecipitation Protocol to Identify Protein-RNA Interactions in Arabidopsis thaliana.

The role of RNA-binding proteins in the regulation of epigenetic processes has received increasing attention in the past decades. In particular noncoding RNAs have been shown to play a role in chromatin loop formation, recruitment of chromatin modifiers and RNA-dependent DNA methylation. In plants, the identification of specific RNA-protein interactions is now rising, facilitated by the development of specific approaches for plant tissues. Here, we present a simple one-day RNA immunoprecipitation (RIP) protocol adapted for Arabidopsis, suited for the identification of RNAs that are associated with a protein-of-interest in planta.

Abstract A11: RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

Abstract The retinoblastoma (RB) tumor suppressor is widely recognized as a master regulator of the transcriptional program that controls entry into the S phase of the cell cycle. Its loss, which is a hallmark of cancer, leads to uncontrolled cell proliferation. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers and remodelers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double strand breaks (DSBs) dependent on E2F1 and ATM kinase activity. RB promotes DNA end resection and DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1 and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability and renders mice hypersensitive to IR. Importantly, depletion of either RB or BRG1 in osteosarcoma and breast cancer cell lines results in sensitivity to DNA damaging agents, thus underscoring the importance of these factors in DNA repair. We uncover a novel, non-transcriptional function for RB in HR, which could contribute to genome instability associated with RB loss. Citation Format: Renier Vélez-Cruz, Swarnalatha Manickavinayaham, Anup K. Biswas, Regina W. Clary, David G. Johnson. RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A11.