Circularized Chromosome Conformation Capture Research Articles

Target genes regulated by CLEC16A intronic region associated with common variable immunodeficiency

BackgroundCLEC16A intron 19 has been identified as a candidate locus for common variable immunodeficiency (CVID). ObjectiveThe objective of this study is to elucidate the molecular mechanism by which variants at the CLEC16A intronic locus may contribute to the pathogenesis of CVID. MethodsWe performed fine-mapping of the CLEC16A locus in a CVID cohort, then deleted the candidate functional SNP in T-cell lines by the CRISPR-Cas9 technique and conducted RNA-seq to identify target gene(s). The interactions between the CLEC16A locus and its target genes were identified using Circularized Chromosome Conformation Capture. The transcription factor complexes mediating the chromatin interactions were determined by proteomic approach. The molecular pathways regulated by the CLEC16A locus were examined by RNA-seq and reverse phase protein array. ResultsOur study showed that the CLEC16A locus is an enhancer regulating expression of multiple target genes including a distant gene ATF7IP2 through chromatin interactions. Distinct transcription factor complexes mediate the chromatin interactions in an allele-specific manner. Disruption of the CLEC16A locus affects the AKT signaling pathway, as well as the molecular response of CD4+T cells to immune stimulation. ConclusionThrough multi-omics and targeted experimental approaches, we elucidated the underlying target genes and signaling pathways involved in the genetic association of CLEC16A with CVID, and highlighted plausible molecular targets for developing novel therapeutics.

An intronic enhancer of Cebpa regulates adipocyte differentiation and adipose tissue development via long-range loop formation.

Cebpa is a master transcription factor gene for adipogenesis. However, the mechanisms of enhancer-promoter chromatin interactions controlling Cebpa transcriptional regulation during adipogenic differentiation remain largely unknown. To reveal how the three-dimensional structure of Cebpa changes during adipogenesis, we generated high-resolution chromatin interactions of Cebpa in 3T3-L1 preadipocytes and 3T3-L1 adipocytes using circularized chromosome conformation capture sequencing (4C-seq). We revealed dramatic changes in chromatin interactions and chromatin status at interaction sites during adipogenic differentiation. Based on this, we identified five active enhancers of Cebpa in 3T3-L1 adipocytes through epigenomic data and luciferase reporter assays. Next, epigenetic repression of Cebpa-L1-AD-En2 or -En3 by the dCas9-KRAB system significantly down-regulated Cebpa expression and inhibited adipocyte differentiation. Furthermore, experimental depletion of cohesin decreased the interaction intensity between Cebpa-L1-AD-En2 and the Cebpa promoter and down-regulated Cebpa expression, indicating that long-range chromatin loop formation was mediated by cohesin. Two transcription factors, RXRA and PPARG, synergistically regulate the activity of Cebpa-L1-AD-En2. To test whether Cebpa-L1-AD-En2 plays a role in adipose tissue development, we injected dCas9-KRAB-En2 lentivirus into the inguinal white adipose tissue (iWAT) of mice to suppress the activity of Cebpa-L1-AD-En2. Repression of Cebpa-L1-AD-En2 significantly decreased Cebpa expression and adipocyte size, altered iWAT transcriptome, and affected iWAT development. We identified functional enhancers regulating Cebpa expression and clarified the crucial roles of Cebpa-L1-AD-En2 and Cebpa promoter interaction in adipocyte differentiation and adipose tissue development.

The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

Mechanotransduction-induced glycolysis epigenetically regulates a CXCL1-dominant angiocrine signaling program in liver sinusoidal endothelial cells invitro and invivo.

Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling invitro and invivo. Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin β1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

Exploring high-resolution chromatin interaction changes and functional enhancers of myogenic marker genes during myogenic differentiation

Skeletal muscle differentiation (myogenesis) is a complex and highly coordinated biological process regulated by a series of myogenic marker genes. Chromatin interactions between gene’s promoters and their enhancers have an important role in transcriptional control. However, the high-resolution chromatin interactions of myogenic genes and their functional enhancers during myogenesis remain largely unclear. Here, we used circularized chromosome conformation capture coupled with next generation sequencing (4C-seq) to investigate eight myogenic marker genes in C2C12 myoblasts (C2C12-MBs) and C2C12 myotubes (C2C12-MTs). We revealed dynamic chromatin interactions of these marker genes during differentiation and identified 163 and 314 significant interaction sites (SISs) in C2C12-MBs and C2C12-MTs, respectively. The interacting genes of SISs in C2C12-MTs were mainly involved in muscle development, and histone modifications of the SISs changed during differentiation. Through functional genomic screening, we also identified 25 and 41 putative active enhancers in C2C12-MBs and C2C12-MTs, respectively. Using luciferase reporter assays for putative enhancers of Myog and Myh3, we identified eight activating enhancers. Furthermore, dCas9-KRAB epigenome editing and RNA-Seq revealed a role for Myog enhancers in the regulation of Myog expression and myogenic differentiation in the native genomic context. Taken together, this study lays the groundwork for understanding 3D chromatin interaction changes of myogenic genes during myogenesis and provides insights that contribute to our understanding of the role of enhancers in regulating myogenesis.

Regulation of MYB by distal enhancer elements in human myeloid leukemia

MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of leukemia. Although the transcription of MYB has been well studied, its detailed underlying regulatory mechanisms still remain elusive. Here, we detected the long-range interaction between the upstream regions, −34k and −88k, and the MYB promoter in K562, U937, and HL-60 cells using circularized chromosome conformation capture (4C) assay, which declined when MYB was downregulated during chemical-induced differentiation. The enrichment of enhancer markers, H3K4me1 and H3K27ac, and enhancer activity at the −34k and −88k regions were confirmed by ChIP-qPCR and luciferase assay respectively. ChIP-qPCR showed the dynamic binding of GATA1, TAL1, and CCAAT/enhancer-binding protein (C/EBPβ) at −34k and −88k during differentiation of K562 cells. Epigenome editing by a CRISPR-Cas9-based method showed that H3K27ac at −34k enhanced TF binding and MYB expression, while DNA methylation inhibited MYB expression. Taken together, our data revealed that enhancer elements at −34k are required for MYB expression, TF binding, and epigenetic modification are closely involved in this process in human myeloid leukemia cells.

Functional Assessment of Coding and Regulatory Variants From the DKK1 Locus

ABSTRACTThe DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome‐wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt‐pathway‐specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high‐bone‐mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture‐ (4C‐) sequencing experiment, we have detected that the region containing 24 BMD‐GWA variants, located 350‐kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA‐activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss‐of‐function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long‐range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett like characteristics.

Myocyte enhancer factor 2C (MEF2C) is a core transcription factor in neurodevelopment and aberrations are typically associated with a Rett-like syndrome, featured by severe intellectual disability, seizures and stereotypic movements. However, structural variants upstream of MEF2C have been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements represents the underlying genetic cause in these cases. To shed light on how these aberrations impact MEF2C regulation, we dissected the MEF2C regulatory landscape. Using Circularized Chromosome Conformation Capture (4C) sequencing in a neuronal cell line, we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers located upstream of the coding sequence. Luciferase reporter assays confirmed enhancer activity for thirteen out of sixteen selected candidate enhancers. Moreover, using enhancer assays in zebrafish, we characterized eight enhancers with in vivo neuronal activity. While each of these enhancers displayed a distinct activity pattern, several showed overlapping activity in the forebrain, notochord or neurons above the eye. In summary, we disentangled a complex regulatory network governing neuronal MEF2C transcription that involves multiple distal enhancers. Disrupting this regulatory structure is likely detrimental to normal neurodevelopment and can give rise to neurodevelopmental disorders such as Rett-like syndrome.

The Atherosclerosis Risk Variant rs2107595 Mediates Allele-Specific Transcriptional Regulation of HDAC9 via E2F3 and Rb1.

Background and Purpose- Genome-wide association studies have identified the HDAC9 (histone deacetylase 9) gene region as a major risk locus for atherosclerotic stroke and coronary artery disease in humans. Previous results suggest a role of altered HDAC9 expression levels as the underlying disease mechanism. rs2107595, the lead single nucleotide polymorphism for stroke and coronary artery disease resides in noncoding DNA and colocalizes with histone modification marks suggestive of enhancer elements. Methods- To determine the mechanisms by which genetic variation at rs2107595 regulates HDAC9 expression and thus vascular risk we employed targeted resequencing, proteome-wide search for allele-specific nuclear binding partners, chromatin immunoprecipitation, genome-editing, reporter assays, circularized chromosome conformation capture, and gain- and loss-of-function experiments in cultured human cell lines and primary immune cells. Results- Targeted resequencing of the HDAC9 locus in patients with atherosclerotic stroke and controls supported candidacy of rs2107595 as the causative single nucleotide polymorphism. A proteomic search for nuclear binding partners revealed preferential binding of the E2F3/TFDP1/Rb1 complex (E2F transcription factor 3/transcription factor Dp-1/Retinoblastoma 1) to the rs2107595 common allele, consistent with the disruption of an E2F3 consensus site by the risk allele. Gain- and loss-of-function studies showed a regulatory effect of E2F/Rb proteins on HDAC9 expression. Compared with the common allele, the rs2107595 risk allele exhibited higher transcriptional capacity in luciferase assays and was associated with higher HDAC9 mRNA levels in primary macrophages and genome-edited Jurkat cells. Circularized chromosome conformation capture revealed a genomic interaction of the rs2107595 region with the HDAC9 promoter, which was stronger for the common allele as was the in vivo interaction with E2F3 and Rb1 determined by chromatin immunoprecipitation. Gain-of-function experiments in isogenic Jurkat cells demonstrated a key role of E2F3 in mediating rs2107595-dependent transcriptional regulation of HDAC9. Conclusions- Collectively, our findings imply allele-specific transcriptional regulation of HDAC9 via E2F3 and Rb1 as a major mechanism mediating vascular risk at rs2107595.

A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett-like characteristics

Mutations in myocyte enhancer factor 2C (MEF2C), an important transcription factor in neurodevelopment, are associated with a Rett-like syndrome. Structural variants (SVs) upstream of MEF2C, which do not disrupt the gene itself, have also been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements can also cause a Rett-like phenotype. To characterize those elements that regulate MEF2C during neural development and that are affected by these SVs, we used genomic tools coupled with both in vitro and in vivo functional assays. Through circularized chromosome conformation capture sequencing (4C-seq) and the assay for transposase-accessible chromatin using sequencing (ATAC-seq), we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers that are deleted or translocated by disease-associated SVs. A total of 16 selected candidate regulatory sequences were tested for enhancer activity in vitro, with 14 found to be functional enhancers. Further analyses of their in vivo activity in zebrafish showed that each of these enhancers has a distinct activity pattern during development, with eight enhancers displaying neuronal activity. In summary, our results disentangle a complex regulatory network governing neuronal MEF2C expression that involves multiple distal enhancers. In addition, the characterized neuronal enhancers pose as novel candidates to screen for mutations in neurodevelopmental disorders, such as Rett-like syndrome.

Cutting Edge: Proper Orientation of CTCF Sites in Cer Is Required for Normal Jκ-Distal and Jκ-Proximal Vκ Gene Usage.

Igκ locus contraction and Vκ gene usage are controlled by Cer, a cis-acting sequence in the Vκ-Jκ intervening region. This effect is attributed to two CTCF-binding sites within Cer that are oriented toward the Vκ gene region. However, the importance of Cer CTCF orientation in regulating VκJκ rearrangement is unknown. We used CRISPR/Cas9 editing to delete and invert Cer in murine Abl pro-B cell lines. This revealed that Cer orientation is critical because clones with either an inverted or deleted Cer element show skewing toward Jκ-proximal Vκ gene usage. However, only Cer deletion increased Jκ-proximal Vκ germline transcription, suggesting an insulating function of Cer. Lastly, circularized chromosome conformation capture interaction data show that Cer CTCF orientation regulates long-range interactions with inversion clones displaying fewer interactions with regions in the middle and distal parts of the Vκ locus and more interactions to downstream regions compared with wild-type or deletion clones.

Insulin promoter in human pancreatic β cells contacts diabetes susceptibility loci and regulates genes affecting insulin metabolism

Both type 1 and type 2 diabetes involve a complex interplay between genetic, epigenetic, and environmental factors. Our laboratory has been interested in the physical interactions, in nuclei of human pancreatic β cells, between the insulin (INS) gene and other genes that are involved in insulin metabolism. We have identified, using Circularized Chromosome Conformation Capture (4C), many physical contacts in a human pancreatic β cell line between the INS promoter on chromosome 11 and sites on most other chromosomes. Many of these contacts are associated with type 1 or type 2 diabetes susceptibility loci. To determine whether physical contact is correlated with an ability of the INS locus to affect expression of these genes, we knock down INS expression by targeting the promoter; 259 genes are either up or down-regulated. Of these, 46 make physical contact with INS We analyze a subset of the contacted genes and show that all are associated with acetylation of histone H3 lysine 27, a marker of actively expressed genes. To demonstrate the usefulness of this approach in revealing regulatory pathways, we identify from among the contacted sites the previously uncharacterized gene SSTR5-AS1 and show that it plays an important role in controlling the effect of somatostatin-28 on insulin secretion. These results are consistent with models in which clustering of genes supports transcriptional activity. This may be a particularly important mechanism in pancreatic β cells and in other cells where a small subset of genes is expressed at high levels.

Cocaine-Induced Chromatin Modifications Associate With Increased Expression and Three-Dimensional Looping of Auts2

BackgroundExposure to drugs of abuse alters the epigenetic landscape of the brain’s reward regions, such as the nucleus accumbens. We investigated how combinations of chromatin modifications affect genes that regulate responses to cocaine. We focused on Auts2, a gene linked to human evolution and cognitive disorders, which displays strong clustering of cocaine-induced chromatin modifications in this brain region. MethodsWe combined chromosome conformation capture, circularized chromosome conformation capture, and related approaches with behavioral paradigms relevant to cocaine phenotypes. Cell type–specific functions were assessed by fluorescence-activated cell sorting and viral-mediated overexpression in Cre-dependent mouse lines. ResultsWe observed that Auts2 gene expression is increased by repeated cocaine administration specifically in D2-type medium spiny neurons in the nucleus accumbens, an effect seen in male but not female mice. Auts2 messenger RNA expression was also upregulated postmortem in the nucleus accumbens of male human cocaine addicts. We obtained evidence that chromosomal looping, bypassing 1524 kb of linear genome, connects Auts2 to the Caln1 gene locus under baseline conditions. This looping was disrupted after repeated cocaine exposure, resulting in increased expression of both genes in D2-type medium spiny neurons. Cocaine exposure reduces binding of CCCTC-binding factor, a chromosomal scaffolding protein, and increases histone and DNA methylation at the Auts–Caln1 loop base in the nucleus accumbens. Cell type–specific overexpression of Auts2 or Caln1 in D2-type medium spiny neurons demonstrated that both genes promote cocaine reward. ConclusionsThese findings suggest that cocaine-induced alterations of neuronal three-dimensional genome organization destabilize higher order chromatin at specific loci that regulate responses to the drug.

PRC2 Facilitates the Regulatory Topology Required for Poised Enhancer Function during Pluripotent Stem Cell Differentiation

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology.

Making Sense of the Tangle: Insights into Chromatin Folding and Gene Regulation.

Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs) that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding. Replication domains are established simultaneously with TADs during the cell cycle and the two correlate well in terms of characteristic features, such as lamin association and histone modifications. CCCTC-binding factor (CTCF) and cohesin cooperate across different cell types to regulate genes and genome organization. CTCF elements that demarcate TAD boundaries are commonly disrupted in cancer and promote oncogene activation. Chromatin looping facilitates interactions between distant promoters and enhancers, and the resulting enhanceosome complex promotes gene expression. Deciphering the chromatin tangle requires comprehensive integrative analyses of DNA- and protein-dependent factors that regulate genomic organization.

4C-seq revealed long-range interactions of a functional enhancer at the 8q24 prostate cancer risk locus.

Genome-wide association studies (GWAS) have identified >100 independent susceptibility loci for prostate cancer, including the hot spot at 8q24. However, how genetic variants at this locus confer disease risk hasn’t been fully characterized. Using circularized chromosome conformation capture (4C) coupled with next-generation sequencing and an enhancer at 8q24 as “bait”, we identified genome-wide partners interacting with this enhancer in cell lines LNCaP and C4-2B. These 4C-identified regions are distributed in open nuclear compartments, featuring active histone marks (H3K4me1, H3K4me2 and H3K27Ac). Transcription factors NKX3-1, FOXA1 and AR (androgen receptor) tend to occupy these 4C regions. We identified genes located at the interacting regions, and found them linked to positive regulation of mesenchymal cell proliferation in LNCaP and C4-2B, and several pathways (TGF beta signaling pathway in LNCaP and p53 pathway in C4-2B). Common genes (e.g. MYC and POU5F1B) were identified in both prostate cancer cell lines. However, each cell line also had exclusive genes (e.g. ELAC2 and PTEN in LNCaP and BRCA2 and ZFHX3 in C4-2B). In addition, BCL-2 identified in C4-2B might contribute to the progression of androgen-refractory prostate cancer. Overall, our work reveals key genes and pathways involved in prostate cancer onset and progression.

Assay for transposase-accessible chromatin and circularized chromosome conformation capture, two methods to explore the regulatory landscapes of genes in zebrafish.

Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome.

NOTCH1 Induces Differential Epigenomic Patterning and Genomic Organization in Fetal Liver- and Adult Bone Marrow-Derived Hematopoietic Progentiors

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T-cell progenitors, characterized by activating NOTCH1 mutations in over 50% of children and adult cases. Although intensive multiagent chemotherapy achieves cure in most pediatric patients, the majority of adults succumb quickly to their disease. The basis for this divergence is likely multifactorial, but we sought in this study to investigate whether cell intrinsic features might contribute to the disparate biologies in pediatric and adult patients.In our prior abstract, we modeled pediatric and adult leukemias by transduction of hematopoietic stem/progenitor cells (HSPC) derived from mouse fetal liver (FL) and adult bone marrow (ABM) with activated NOTCH1 virus followed by transplantation into histocompatible recipient animals. We observed that whereas FL- and ABM-derived HSPC generate similar primary acute T-cell leukemias in terms of penetrance, latency, disease burden/distribution, and immunophenotype, FL leukemias exhibit much greater cycling activity than ABM leukemias, yet are dramatically impaired in their ability to propagate disease in secondary and tertiary recipients compared to ABM leukemias. Using a combination of gene expression profiling and in vitro culture assays, we attributed this differential behavior to NOTCH1-induced autocrine IGF signaling that is operative in FL, but not ABM-derived HSPC.Here we report that NOTCH1 mediates its effects on IGF1 in FL-derived HSPC directly by physical occupancy over the IGF1 promoter in a dimerization-dependent fashion. As well, increased NOTCH1 occupancy at the IGF1 promoter region in FL tissues is associated with reduced histone H3K27 trimethylation (a mark of transcriptionally silent chromatin), yet there is equivalent histone H3K4 trimethylation (a mark identifying transcriptionally active promoters) in both FL and ABM tissues, suggesting that NOTCH1 may be responsible for interconverting the IGF1 locus between active and inactive, but poised chromatin states. NOTCH1 occupancy is also associated with enhanced physical interactions between the IGF1 promoter region and distant genomic loci as revealed by circularized chromosome conformation capture (4C) assay and confirmed by chromosome conformation capture (3C) assay, including sites with H3K4 monomethylation (a mark of transcriptional enhancers) suggesting that NOTCH1 promotes "looping in" of distant enhancer elements that drive IGF1 expression in FL tissues. We conclude from these studies that NOTCH1 enacts differential, developmental stage-specific transcriptional programs by a combination of local epigenetic patterning and long-range genomic interactions. These findings support the notion that pediatric and adult T-ALL may potentially be regarded as related, but biologically distinct diseases, and that novel, age-specific therapies that exploit these differences may improve clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

Analysis of a transgenic Oct4 enhancer reveals high fidelity long-range chromosomal interactions.

Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture (4C) coupled with next generation sequencing, we determined and compared the genome-wide interactome of the endogenous and transgenic Oct4 distal enhancers. Integrative genomic analysis indicated that the transgenic enhancer binds to a similar set of loci and shares similar key enrichment profiles with its endogenous counterpart. Both the endogenous and transgenic Oct4 enhancer interacting loci were enriched in the open nucleus compartment, which is associated with active histone marks (H3K4me1, H3K27ac, H3K4me3 and H3K9ac), active cis-regulatory sequences (DNA hypersensitivity sites (DHS)), 5-hydroxymethylcytosine (5-hmc), and early DNA replication domains. In addition, binding of some pluripotency-related transcription factors was consistently enriched in our 4C sites, and genes in those sites were generally more highly expressed. Overall, our work reveals critical features that may function in gene expression regulation in mouse pluripotent cells.

Abstract A31: Elucidating mechanisms that cooperate with the therapeutic inhibition of RNA polymerase I to treat MYC-driven hematological malignancies

Abstract The MYC oncoprotein and transcription factor has a well-described role in harnessing and driving ribosome biogenesis (RiBi) via its regulation of all three RNA polymerases, and it is proposed that this is a crucial output of MYC's malignant function. Thus, the potential for therapeutic intervention in a broad range of MYC-driven cancers may converge on targeting the RiBi program, potentially as monotherapy or, more likely, as a basis for rational combination therapies. Indeed, small molecule (CX-5461) inhibition of RNA Polymerase I (Pol I), a major MYC target, selectively kills MYC-driven lymphoma in vivo via activation of a p53-dependent nucleolar stress response. While CX-5461 potently inhibits ribosome biogenesis in a MYC-driven B-cell lymphoma via p53-dependent apoptosis, it is unclear whether all malignancies will respond similarly. Furthermore, as we predict resistance to CX-5461 and tumor relapse from our in vivo mouse studies, the need to identify pathways responsible for mediating resistance to Pol I inhibition in the background of oncogenic MYC is necessary for future development of rational combination therapies. To address these key questions, we have undertaken multiple approaches. Firstly, we are utilizing model systems of acute myeloid leukemia (AML) to take both an unbiased and a direct approach to understanding the role of MYC in mediating CX-5461 sensitivity. Secondly, to identify factors conferring acquired resistance to CX-5461 we have used the well-characterized Eµ-Myc mouse model of MYC-mediated lymphoma where, following a period of disease remission, prolonged dosing of CX-5461 in mice bearing lymphomas results in eventual relapse due to acquired drug resistance. Thus, we compared whole-exome sequencing data between isogenic sensitive and CX-5461 resistant B-cell lymphomas to identify additional mutations conferring drug resistance. Finally, we are using the Eµ-Myc model to investigate the regulation of rDNA transcription itself during cancer progression. Compared to wild type B-cells, malignant MYC over-expressing B-cells have hyper-activated Pol I and increased rDNA transcription. This increase in transcription is associated with a dramatic increase in the number of transcriptionally active rDNA repeats, indicating changes in chromatin structure during transformation. In order to assess epigenetic changes at the rDNA loci and to identify long distance rDNA interactions occurring during malignant progression, we have used ChIP-seq and circularized chromosome conformation capture sequencing (4C-seq) at all cell stages of malignancy in vivo (wild type, pre-malignant and malignant Eµ-Myc B-cells). We demonstrate that transition from premalignancy to malignancy is associated with robust changes in rDNA chromatin as well as reorganization of rDNA-genome interactions with a significant increase in these interactions being detected in malignant cells. Our data provide evidence for MYC-driven activation of rDNA in cancer progression beyond ribosome biogenesis and offer novel insights into the spatial and transcriptional dynamics of the rDNA-associated genome during malignant transformation. We will present our latest data on all fronts aimed at understanding the critical role that MYC plays in therapeutic inhibition of Pol I transcription. Citation Format: Gretchen Poortinga, Jeannine Diesch, Elaine Sanij, Nadine Hein, Jirawas Sornkum, Donald Cameron, Megan J. Bywater, Ricky Johnstone, Denis Drygin, Sean O'Brien, Richard B. Pearson, Grant A. McArthur, Ross D. Hannan. Elucidating mechanisms that cooperate with the therapeutic inhibition of RNA polymerase I to treat MYC-driven hematological malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A31.