Direct DNA Binding Research Articles

Direct DNA binding by BRCA1 on β-hCG promoter and its clinical implications

ObjectiveThe role of β-hCG in breast cancer is largely unknown, this study aims to analyse the gene expression and clinical implications of β-hCG and its isoforms in various cancers focussing particularly in Breast Invasive Carcinoma (BRCA). A mechanistic approach deciphering the transcriptional regulation of β-hCG by BRCA1 was also explored. MethodsData from various comprehensive gene expression platforms like UALCAN, GEPIA2, GENT2, TIMER2, LinkedOmics, and STRING were used to analyse the expression of β-hCG and its clinical implications; Immunohistochemistry and ELISA for β-hCG expression analysis from human breast cancer patients; Electrophoretic mobility shift assay (EMSA) to analyse the direct binding of BRCA1 on β-hCG; Immunoblotting and Luciferase assay to understand the regulation of β-hCG by p53 were performed. ResultsResults from UALCAN and GENT2 gene expression cancer database revealed that TNBC subtypes and high-grade metaplastic carcinoma shows elevated expression of β-hCG and infiltration of various immune cells were also identified in BRCA by TIMER2. It was observed that most of the isoforms of β-hCG (CGB) are upregulated in breast cancers irrespective of hormonal status when BRCA1 gene is mutated according to TIMER2. Similar results were observed with Lymphoid neoplasm diffuse large B-cell lymphoma (LGG) and DLBC (Brain lower grade glioma) when BRCA1 is mutated. These results correlate with our earlier reports indicating expression of β-hCG in BRCA1 defective condition. We have also identified direct binding of BRCA1 on β-hCG promoter. ConclusionAll these findings demonstrate the importance of β-hCG as a potential target in BRCA1-deficient carcinomas.

Targeting pericentric non-consecutive motifs for heterochromatin initiation.

Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1-3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.

Transcription factor OsWRKY11 induces rice heading at low concentrations but inhibits rice heading at high concentrations.

The heading date of rice is a crucial agronomic characteristic that influences its adaptability to different regions and its productivity potential. Despite the involvement of WRKY transcription factors in various biological processes related to development, the precise mechanisms through which these transcription factors regulate the heading date in rice have not been well elucidated. The present study identified OsWRKY11 as a WRKY transcription factor which exhibits a pivotal function in the regulation of the heading date in rice through a comprehensive screening of a clustered regularly interspaced palindromic repeats (CRISPR) ‒ CRISPR-associated nuclease 9 mutant library that specifically targets the WRKY genes in rice. The heading date of oswrky11 mutant plants and OsWRKY11-overexpressing plants was delayed compared with that of the wild-type plants under short-day and long-day conditions. Mechanistic investigation revealed that OsWRKY11 exerts dual effects on transcriptional promotion and suppression through direct and indirect DNA binding, respectively. Under normal conditions, OsWRKY11 facilitates flowering by directly inducing the expression of OsMADS14 and OsMADS15. The presence of elevated levels of OsWRKY11 protein promote formation of a ternary protein complex involving OsWRKY11, Heading date 1 (Hd1), and Days to heading date 8 (DTH8), and this complex then suppresses the expression of Ehd1, which leads to a delay in the heading date. Subsequent investigation revealed that a mild drought condition resulted in a modest increase in OsWRKY11 expression, promoting heading. Conversely, under severe drought conditions, a significant upregulation of OsWRKY11 led to the suppression of Ehd1 expression, ultimately causing a delay in heading date. Our findings uncover a previously unacknowledged mechanism through which the transcription factor OsWRKY11 exerts a dual impact on the heading date by directly and indirectly binding to the promoters of target genes.

Using i-GONAD for Cell-Type-Specific and Systematic Analysis of Developmental Transcription Factors In Vivo.

Transcription factors (TFs) regulate gene expression via direct DNA binding together with cofactors and in chromatin remodeling complexes. Their function is thus regulated in a spatiotemporal and cell-type-specific manner. To analyze the functions of TFs in a cell-type-specific context, genome-wide DNA binding, as well as the identification of interacting proteins, is required. We used i-GONAD (improved genome editing via oviductal nucleic acids delivery) in mice to genetically modify TFs by adding fluorescent reporter and affinity tags that can be exploited for the imaging and enrichment of target cells as well as chromatin immunoprecipitation and pull-down assays. As proof-of-principle, we showed the functional genetic modification of the closely related developmental TFs, Bcl11a and Bcl11b, in defined cell types of newborn mice. i-GONAD is a highly efficient procedure for modifying TF-encoding genes via the integration of small insertions, such as reporter and affinity tags. The novel Bcl11a and Bcl11b mouse lines, described in this study, will be used to improve our understanding of the Bcl11 family's function in neurodevelopment and associated disease.

P53 governs an AT1 differentiation programme in lung cancer suppression.

Lung cancer is the leading cause of cancer deaths worldwide1. Mutations in the tumour suppressor gene TP53 occur in 50% of lung adenocarcinomas (LUADs) and are linked to poor prognosis1-4, but how p53 suppresses LUAD development remains enigmatic. We show here that p53 suppresses LUAD by governing cell state, specifically by promoting alveolar type 1 (AT1) differentiation. Using mice that express oncogenic Kras and null, wild-type or hypermorphic Trp53 alleles in alveolar type 2 (AT2) cells, we observed graded effects of p53 on LUAD initiation and progression. RNA sequencing and ATAC sequencing of LUAD cells uncovered a p53-induced AT1 differentiation programme during tumour suppression in vivo through direct DNA binding, chromatin remodelling and inductionof genes characteristic of AT1 cells. Single-cell transcriptomics analyses revealed that during LUAD evolution, p53 promotes AT1 differentiation throughaction in a transitional cell state analogous to a transient intermediary seen during AT2-to-AT1 cell differentiation in alveolar injury repair. Notably, p53 inactivation results in the inappropriate persistence of these transitional cancer cells accompanied by upregulated growth signalling and divergence from lung lineage identity, characteristics associated with LUAD progression. Analysis of Trp53 wild-type and Trp53-null mice showed that p53 also directs alveolar regeneration after injury by regulating AT2 cell self-renewal and promoting transitional cell differentiation into AT1 cells. Collectively, these findings illuminate mechanisms of p53-mediated LUAD suppression, in which p53 governs alveolar differentiation, and suggest that tumour suppression reflects a fundamental role of p53 in orchestrating tissue repair after injury.

Murine CD4+ T cells exhibit sexually dimorphic responses to estrogen signaling

Abstract Many autoimmune diseases exhibit sexual dimorphism. 17β-estradiol (estrogen, “E2”), a steroid sex hormone primarily known for its reproductive roles, has also been shown to modulate CD4+ T cells. E2 signals through two nuclear receptors, ERα and ERβ, which regulate gene transcription through direct DNA binding and other mechanisms. We previously showed that in the SAMP/YitFC model of Crohn’s-like ileitis, loss of ERβ enhanced inflammation selectively in female mice. We also found that loss of ERβ resulted in decreased expression of Foxp3 in CD4+ T cells, suggesting that ERβ is required for differentiation of Foxp3+ Tregs. These prior findings suggest a pro-inflammatory role for ERα and anti-inflammatory role for ERβ, potentially in a sex-specific manner. In the current study, we investigated mechanisms of E2 signaling in CD4+ T cells isolated from males vs. females. Using standard in vitro assays, we found that the proliferation of TGFβ-induced regulatory T cells (Tregs) was enhanced in female cells co-treated with E2 compared to male cells, suggesting that E2 enhances Treg differentiation more strongly in females. Interestingly, using the CD45RB T cell transfer model of colitis, we found that recipients of female donor cells exhibited more severe disease compared to recipients of male cells, suggesting that female-derived CD45RB highT cells are more colitogenic than male cells. Future studies will determine the effect of E2 signaling on both effector and regulatory CD4+ T cell populations, identifying the contributions of ERα vs. ERβ-specific signaling in these T cell subsets and the functional impact of these signaling pathways in males versus females. Supported by grants from NIH (R01DK128143, R03DK123579, T32AI089474)

Strategies for the Voltammetric Detection of Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is rapidly developing into an important tool for the point-of-use detection of pathogens for both clinical and environmental samples, largely due to its sensitivity, rapidity, and adaptability to portable devices. Many methods are used to monitor LAMP, but not all are amenable to point-of-use applications. Common methods such as fluorescence often require bulky equipment, whereas colorimetric and turbidimetric methods can lack sensitivity. Electrochemical biosensors are becoming increasingly important for these applications due to their potential for low cost, high sensitivity, and capacity for miniaturization into integrated devices. This review provides an overview of the use of voltammetric sensors for monitoring LAMP, with a specific focus on how electroactive species are used to interface between the biochemical products of the LAMP reaction and the voltammetric sensor. Various strategies for the voltammetric detection of DNA amplicons as well as pyrophosphate and protons released during LAMP are presented, ranging from direct DNA binding by electroactive species to the creative use of pyrophosphate-detecting aptamers and pH-sensitive oligonucleotide structures. Hurdles for adapting these devices to point-of-use applications are also discussed.

BPDCN MYB Fusions Bind Cell Cycle Gene Promoters, Reactivate Self-Renewal in Progenitor Cells and Induce Myeloid/Dendritic Leukemia

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare acute leukemia with poor prognosis arising from the plasmacytoid dendritic cell (pDC) lineage. MYB is a hematopoietic transcription factor overexpressed in many leukemias. Genomic rearrangements of MYB were identified in a significant fraction of BPDCNs, including in cases without any other clear driver mutations, and result in its fusion to one of several partner genes (Suzuki et al., Leukemia 2017). These fusions are rare or absent in other acute leukemias and the mechanisms by which they contribute to BPDCN oncogenesis are not understood. To examine the impact of Myb fusions on hematopoietic differentiation and leukemogenesis, we used mouse bone marrow progenitors immortalized by estradiol (E2)-dependent activation of HoxB8 and culture with Flt3 ligand (HoxB8-FL cells). We used HoxB8-FL cells deficient in Cdkn2a, since CDKN2A deletion is present in up to ~67% of BPDCNs (Lucioni et al., Blood 2011) but is rare in other myeloid leukemias. HoxB8-FL cells differentiate into myeloid, lymphoid and dendritic cells in vitro upon withdrawal of E2, or in vivo upon transplantation. They therefore represent lymphoid-primed multipotent progenitor (LMPP)-like cells that are unable to self-renew in the absence of E2. We co-expressed a dTom reporter with V5-tagged Myb constructs in HoxB8-FL cells: wild-type full-length Myb (Myb-FL), truncated Myb (Myb-TR) or Myb-PLEKHO1. MYB-PLEKHO1 is the most frequent fusion in BPDCN patients, while truncated Myb is analogous to recurrent BPDCN MYB fusions in which the partner is out-of-frame. Empty vector was used as a control. HoxB8-FL cells expressing Myb-FL, Myb-TR or Myb-PLEKHO1 displayed arrested differentiation upon withdrawal of E2 in vitro (Figure 1). While all empty vector-transduced cells had upregulated CD11b and/or CD11c at day 7 post-E2 withdrawal, Myb overexpression caused an accumulation of CD11b-CD11c- undifferentiated cells. In vivo, a similar effect was observed at day 7 post-transplantation: dTom+ CD11b-CD11c-CD19-B220- undifferentiated cells were still present in the bone marrow of Myb-FL, Myb-TR and Myb-PLEKHO1 recipient mice, while in empty vector recipients all dTom+ cells expressed at least one of these differentiation markers. Surprisingly, we observed long-term persistence of dTom+ cells in the peripheral blood of Myb-TR and Myb-PLEKHO1, but not Myb-FL, recipients. This developed into lethal malignancy in 100% of Myb-TR and Myb-PLEKHO1 recipients by 28 weeks (Figure 2). Malignant cells showed blast-like morphology of acute leukemia and expressed markers of a myeloid/dendritic progenitor (Kit+CD11b+Cx3cr1+). dTom+ disease was never observed in Myb-FL or empty vector recipients (log-rank P<0.001). Together, while expression of either full-length Myb or Myb fusions impaired myeloid/dendritic differentiation, only Myb rearrangements associated with BPDCN generated a fully penetrant leukemia. To investigate chromatin-level differences between physiologically expressed wild-type MYB and MYB fusions, we performed Cut&Run in K562 cells with V5-tagged MYB-FL, MYB-TR or MYB-PLEKHO1 knocked in to the endogenous MYB locus using CRISPR/Cas9 and homology-directed repair. We observed increased binding of MYB-TR and MYB-PLEKHO1 relative to MYB-FL at promoters of cell cycle genes that function at the G2/M checkpoint, including CDC20, CDCA3 and CKS2. These binding sites contained canonical MYB binding motifs, indicating that MYB fusions increase direct DNA binding at chromatin involved in control of specific cell cycle genes. Cut&Run for V5-Myb-PLEKHO1 in mouse leukemias revealed similar direct DNA binding at cell cycle gene promoters. Moreover, expression of G2/M cell cycle genes was enriched in patient BPDCNs relative to normal pDCs. Our data suggest that BPDCN MYB fusions exert distinct oncogenic effects to overexpression of wild-type MYB. In addition to arresting differentiation, BPDCN MYB fusions can reactivate self-renewal in progenitor cells, which may occur through increased direct DNA binding at cell cycle gene promoters. This may explain the simple genetics of MYB-rearranged BPDCN, which lack other recurrent myeloid-type mutations, if the fusions are sufficient to generate leukemia via these dual roles. These data also indicate that therapeutic strategies targeting MYB and/or specific cell cycle checkpoints may be active in BPDCN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Comprehensive Reassessment of the Direct DNA Binding Sites of PML-Rara within the Genomes of Primary Hematopoietic Progenitor Cells, and Their Effects on Chromatin Structure and Gene Expression

A Comprehensive Reassessment of the Direct DNA Binding Sites of PML-Rara within the Genomes of Primary Hematopoietic Progenitor Cells, and Their Effects on Chromatin Structure and Gene Expression

OR30-3 Direct and VGLL1-Mediated Gene Regulation by GRHL2 During Mesenchymal to Epithelial Transition in Breast Cancer

Abstract The transcription factor (TF) GRHL2 controls normal epithelial differentiation in different tissues. It is expressed in all breast cancer subtypes except those with a mesenchymal phenotype (claudin-low subtype), where its ectopic expression leads to induction of a mesenchymal-epithelial transition (MET) (1). Here we explored the mechanisms by which GRHL2 induces MET in claudin-low breast cancer cell lines using a combination of ChIP-seq and RNAseq analyses upon modulation of GRHL2 expression in mesenchymal and epithelial cell lines. Direct DNA binding by GRHL2 was enriched in proximity of genes positively regulated in mesenchymal cell lines, which include several epithelial TFs as well as an array of epithelial genes. Biotinylation and mass spectrometry analysis of membrane proteins confirmed surfaceome remodeling via induction of membrane proteins with roles in cell-cell contacts and adhesion. Repression of mesenchymal genes was mediated in part by direct induction of miRNAs targeting mesenchymal transcription factors. In addition, GRHL2 strongly induced expression of the cofactor VGLL1, whose expression is high in the triple-negative (TNBC) basal-like subtype. VGLL1 overexpression in mesenchymal cell lines and suppression in basal-like TNBC cells confirmed its role in mediating part of GRHL2 signaling, in particular via interaction with and modulation of TEAD transcription factors. In addition, GRHL2 also interacted with VGLL1 upon its induction in mesenchymal cells, recruiting it to some of its binding motifs, suggesting a multilayered cross-talk between GRHL2 and TEAD factors. Together, our results indicate that GRHL2 acts as a master regulator in the induction of TNBC epithelial differentiation via direct upregulation of epithelial transcription factors, miRNAs and TEAD cofactor VGLL1, resulting in both upregulation of epithelial genes and suppression of mesenchymal genes.(1) Cieply B. et al., Cancer Res. 2013 Oct. 1;73 (20): 6299-309 Presentation: Tuesday, June 14, 2022 10:15 a.m. - 10:30 a.m.

NMR resonance assignments of the DNA binding domain of mouse Junctophilin-2.

Junctophilin-2 (JP2) is a critical structural protein in the heart by stabilizing junctional membrane complexes between the plasma membrane and sarcoplasmic reticula responsible for precise Ca2+ regulation. Such complexes are essential for efficient cardiomyocyte contraction and adaptation to altered cardiac workload conditions. Mutations in the JPH2 gene that encodes JP2 are associated with inherited cardiomyopathies and arrhythmias, and disruption of JP2 function is lethal. Interestingly, cardiac stress promotes the proteolytic cleavage of JP2 that triggers the translocation of its N-terminal fragment into the nucleus to repress maladaptive gene transcription. We previously found that the central region of JP2 is responsible for mediating direct DNA binding interactions. Recent structural studies indicate that this region serves as a structural role in the cytosolic form of JP2 by folding into a single continuous α-helix. However, the structural basis of how this DNA-binding domain interacts with DNA is not known. Here, we report the backbone and sidechain assignments of the DNA-binding domain (residues 331-413) of mouse JP2. These assignments reveal that the JP2 DNA binding domain is an intrinsically disordered protein and contains two α-helices located in the C-terminal portion of the protein. Moreover, this protein binds to DNA in a similar manner to that shown previously by electrophoretic mobility shift assays. Therefore, these assignments provide a framework for further structural studies into the interaction of this JP2 domain with DNA for the elucidation of transcriptional regulation of stress-responsive genes as well as its role in the stabilization of junctional membrane complexes.

A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression

Used widely for genome editing, CRISPR-Cas enzymes provide RNA-guided immunity to microbes by targeting foreign nucleic acids for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. These crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Nevertheless, Cas12c represses transcription and can defend bacteria against lytic bacteriophage infection when targeting an essential phage gene. Together, these results show that Cas12c employs targeted DNA binding to provide antiviral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.

Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis

Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which—depending on the cellular context—can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain–associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27kip1 and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO–TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.

Telomere length regulation by Rif1 protein from Hansenula polymorpha.

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism - from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension - a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

Post-transcriptional regulation of factors important for the germ line.

Echinoderms are a major model system for many general aspects of biology, including mechanisms of gene regulation. Analysis of transcriptional regulation (Gene regulatory networks, direct DNA-binding of proteins to specific cis-elements, and transgenesis) has contributed to our understanding of how an embryo works. This chapter looks at post-transcriptional gene regulation in the context of how the primordial germ cells are formed, and how the factors essential for this process are regulated. Important in echinoderms, as in many embryos, is that key steps of fate determination are made post-transcriptionally. This chapter highlights these steps uncovered in sea urchins and sea stars, and links them to a general theme of how the germ line may regulate its fate differently than many of the embryo's somatic cell lineages.

Importin α2 association with chromatin: Direct DNA binding via a novel DNA-binding domain.

The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain)-in the N-terminal region of importin α within the conventional importin β binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.

P53 mediates target gene association with nuclear speckles for amplified RNA expression

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response.

ATF6 is a member of the basic-leucine zipper family of transcription factors. It contains a transmembrane domain and is located in membranes of the endoplasmic reticulum. ATF6 has been implicated in the endoplasmic reticulum (ER) stress response pathway since it can activate expression of GRP78 and other genes induced by the ER stress response. ER stress appears to activate ATF6 by cleavage from the ER membrane and translocation to the nucleus. However, direct DNA binding by ATF6 had not been demonstrated. In this report, we have identified a consensus DNA binding sequence for ATF6. This site is related to but distinct from ATF1/CREB binding sites. The site was placed in a reporter gene and was specifically activated by ATF6 overexpression and was strongly induced by the ER stress response. A dominant negative form of ATF6 blocked ER stress induction of both ATF6 site and GRP78 reporter genes. We further found that GAL4-ATF6 could be activated by ER stress. These results demonstrate that ATF6 is a direct target of the ER stress response. A proximal sensor of the ER stress response, human IRE1 (hIRE1), was sufficient to activate the ATF6 reporter gene, while a dominant negative form of hIRE1 blocked ER stress activation, suggesting that hIRE1 is upstream of ATF6 in the ER stress signaling pathway.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram‐positive bacteria. Most functional assessments of CcpA, including interaction with its key co‐factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA‐mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP‐seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL‐8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221‐CcpA‐V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP‐seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild‐type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr‐dependent and independent activities of CcpA in a key pathogenic bacterium.

DIPG-43. CAN WE REPROGRAM DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)? EXPLORING THE ROLE OF DISTALLESS/DLX HOMEOBOX GENE REGULATION OF OLIGODENDROGLIAL PROGENITOR CELLS (OPC) IN THE DEVELOPING VERTEBRATE NERVOUS SYSTEM

BACKGROUNDThe identification of H3.3/H3.1K27M in most DIPG has changed our understanding of this disease. H3K27M mutations usually demonstrate global loss of H3K27 trimethylation (me3) with gain of H3K27 acetylation (ac). Single cell RNAseq has identified the putative cell of origin as oligodendroglial progenitor cells (OPC). The distalless gene family is necessary for the differentiation and tangential migration of committed neural progenitors to become GABAergic interneurons. Dlx1/Dlx2 double knockout (DKO) cells from the ganglionic eminences (GE) transplanted into a wild-type environment become oligodendrocytes.RESULTSWe identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo and confirmed direct DLX2 protein-promoter DNA binding in vitro. Co-expression of Dlx2 with target sequences reduced reporter gene expression in vitro. There was increased expression of OLIG2, NKX2.2 and PLP-1 expression in vivo, consistent with de-repression in the absence of Dlx1/Dlx2 function. Transient over-expression of a Dlx2-GFP construct into murine DIPG cells from a GEMM that develops DIPG resulted in significant increases in expression of Gad isoforms with concomitant decreases in Olig2 and Nkx2.2. Dlx2-transfected mDIPG cells also demonstrated reduced migration, invasion and colony formation in vitro. Of significance, there was global restoration of H3K27me3 with corresponding loss of H3K27ac expression in transfected cells compared to controls.CONCLUSIONSDLX2 promotes GABAergic differentiation and migration while concomitantly repressing OPC differentiation in vivo. Developmental reprogramming of mDIPG cells by DLX2 demonstrates the potential role for directed differentiation strategies towards improving patient outcomes for this devastating pediatric cancer.