Active Chromatin Structure Research Articles

Activation of Hottip lncrna Perturbs HSC Function Leading to AML like Disease in Mice

Several HOX loci associated long noncoding RNAs (lncRNAs) have been shown to regulate transcription of HOX genes through influencing epigenetic landscape. Especially, the posterior HOXA domain associated lncRNA HOTTIP acts as an epigenetic regulator that recruits WDR5/MLL complex to coordinate active chromatin modifications and HOXA genes expression in the development of animal digits. Despite HOX genes, especially HOXA genes, are highly expressed in many acute myeloid leukemia (AML) patients, it remains largely unknown whether and how HOTTIP lncRNA regulates hematopoietic stem cell (HSC) function and contributes to leukemogenesis.We showed previously that disruption of the CTCF boundary located between HOXA7 and HOXA9 genes (CBS7/9) resulted in reduced lncRNA HOTTIP and HOXA genes expression in MLL rearranged AML suggesting that HOTTIP may play a role in ectopic expression of the posterior HOXA gene. We employed a pooled CRISPR-Cas9 KO library to specifically screen lncRNAs in four HOX gene loci and identify HOTTIP as acritical regulator in controlling oncogenic HOX chromatin signature and associated gene expression patterns in AML by collaborating with posterior HOXA chromatin boundary. HOTTIP is upregulated in AML patients with MLL-rearrangement or NPM1 mutation. AML patients with high HOTTIP expression exhibits a significantly shortened survival compared to low HOTTIP expressing patients. To test whether HOTTIP acts to coordinate posterior chromatin domain and HOXA genes activation in AML, we manipulated HOTTIP lncRNA expression levels in the MLL-AF9 rearranged MOLM13 by loss-of-function KO and gain-of function rescue, as well as carried out genome wide chromatin and transcriptomic analysis to intterrogate the role of HOTTIP in control of AML specific posterior HOXA chromatin domain. We found that knock-out of HOTTIP lncRNA led to a loss of active chromatin structure and invasion of repressive H3K27me3 mark over the posterior HOXA domain. HOTTIP KO attenuated progression of AML in the transplanted AML mouse model resembling the effect of CBS7/9 boundary disruption, while transcriptional activation of HOTTIP lncRNA in the CBS7/9 boundary-disrupted AML cells restored HOXA locus chromatin signature and gene expression as well as reversed the CBS7/9-mediated anti-leukemic effects.To further determine the role of HOTTIP lncRNA in regulating HSC function and leukemogenesis, we generated transgenic mice that expresses Hottip lncRNA under the control of the hematopoietic specific Vav1 enhancer and promoter. The Hottip transgenic (Tg) mice exhibited increased WBC and neutrophil counts and developed splenomegaly indicating that enforced expression of Hottip lncRNA resulted in perturbation of hematopoiesis. Furthermore, overexpression of Hottip lncRNA in mice bone marrow hematopoietic compartment strongly perturbed hematopoietic stem and progenitor cell (HSC/HPC) function by altering self-renewal and differentiation property of HSC/HPCs through affecting homeotic gene associated oncogenic transcription program. Approximately 20% of Hottip lncRNA transgenic mice developed abnormal hematopoietic phenotypes resembling AML-like disease. RNA-seq and ATAC-seq analysis indicated that overexpression of Hottip enhanced promoter chromatin accessibility and stimulates transcription of genes and pathways involved in HSC function and leukemogenesis, including WNT signaling, hematopoietic cell lineage, cell cycle, Hoxa9, Hoxa13, and Meis1, Runx1, and Twist1 genes. Thus, Hottip lncRNA overexpression acts as an oncogenic event to promote HSC self-renewal and HPC proliferation by reprograming leukemic associated chromatin signature and transcription programs. DisclosuresNo relevant conflicts of interest to declare.

LRF acts as an activator and repressor of the human β-like globin gene transcription in a developmental stage dependent manner.

Leukemia/lymphoma-related factor (LRF; a hematopoietic transcription factor) has been suggested to repress fetal γ-globin genes in the human adult stage β-globin locus. Here, to study the role of LRF in the fetal stage β-globin locus, we knocked out its expression in erythroid K562 cells, in which the γ-globin genes are mainly transcribed. The γ-globin transcription was reduced in LRF knock-out cells, and transcription factor binding to the β-globin locus control region hypersensitive sites (LCR HSs) and active histone organization in the LCR HSs were disrupted by the depletion of LRF. In contrast, LRF loss in the adult stage β-globin locus did not affect active chromatin structure in the LCR HSs and induced the fetal γ-globin transcription. These results indicate that LRF may act as an activator and repressor of the human β-like globin gene transcription in a manner dependent on developmental stage.

RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Downregulation of PARP1 transcription by promoter-associated E2F4-RBL2-HDAC1-BRM complex contributes to repression of pluripotency stem cell factors in human monocytes

Differentiation of certain cell types is followed by a downregulation of PARP1 expression. We show that the reduction in the abundance of PARP1 in hematopoietic progenitor cells and monocytes is tightly controlled by the cell cycle. The differentiation-associated cell cycle exit induces E2F1 replacement with E2F4 at the PARP1 promoter and the assembly of an E2F4-RBL2-HDAC1-BRM(SWI/SNF) repressor complex which deacetylates nucleosomes and compacts chromatin. In G1 arrested cells, PARP1 transcription is reduced by the recruitment of E2F1-RB1-HDAC1-EZH2(PRC2)-BRM/BRG1(SWI/SNF), which additionally trimethylates H3K27 and causes an even higher increase in nucleosome density. The re-establishment of an active chromatin structure by treating post-mitotic monocytes with the HDAC inhibitor and G1 arrested cells with a combination of HDAC and EZH2 inhibitors restores PARP1 expression completely but does not affect the interaction between the components of the repressor complex with chromatin. This suggests that RB1 and RBL2, as well as PRC2, SWI/SNF and HDAC1, do not interfere with the transcription machinery. Interestingly, reinstatement of PARP1 expression by the silencing of RBL2 or by the inhibition of HDACs in monocytes and by transfection with the PARP1 expression vector in differentiated THP-1 cells substantially increased transcription of pluripotency stem cell factors such as POU5F1, SOX2 and NANOG.

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.

Erythroid specific activator GATA-1-dependent interactions between CTCF sites around the β-globin locus

CTCF sites (binding motifs for CCCTC-binding factor, an insulator protein) are located considerable distances apart on genomes but are closely positioned in organized chromatin. The close positioning of CTCF sites is often cell type or tissue specific. Here we analyzed chromatin organization in eight CTCF sites around the β-globin locus by 3C assay and explored the roles of erythroid specific transcription activator GATA-1 and KLF1 in it. It was found five CTCF sites convergent to the locus interact with each other in erythroid K562 cells but not in non-erythroid 293 cells. The interaction was decreased by depletion of GATA-1 or KLF1. It accompanied reductions of CTCF and Rad21 occupancies and loss of active chromatin structure at the CTCF sites. Furthermore Rad21 occupancy was reduced in the β-globin locus control region (LCR) hypersensitive sites (HSs) by the depletion of GATA-1 or KLF1. The role of GATA-1 in interaction between CTCF sites was revealed by its ectopic expression in 293 cells and by deletion of a GATA-1 site in the LCR HS2. These findings indicate that erythroid specific activator GATA-1 acts at CTCF sites around the β-globin locus to establish tissue-specific chromatin organization.

Whole-Genome Analysis of the Chromatin Structure in Multiple Myeloma

During the last years, the study of molecular alterations associated with multiple myeloma (MM) has been mostly focused on the analysis of the genome, transcriptome and DNA methylome. These analyses are showing that (epi)genetic heterogeneity and extensive perturbation of the transcriptional landscape are hallmarks of MM. Our previous analysis of the whole DNA methylome of MM revealed that this epigenetic mark globally shows a poor correlation with gene expression, and therefore did not allow us to better understand gene deregulation in MM. In contrast, the chromatin structure and histone modifications are emerging as essential epigenetic layers to understand the mechanisms underlying gene expression changes in cancer, but remain widely unexplored in MM.We have now performed a deep ChIP-seq profiling of CD138+ sorted cells from bone marrow samples obtained from four MM patients and three biological replicates of normal plasma cells (NPCs) using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3 and H3K9me3. Different combinations of these marks allow us to segment the MM and NPC genome into functional chromatin states, including active, weak or poised promoters, active or weak enhancers, transcriptional elongation, polycomb-repressed regions and heterochromatic regions.The initial unsupervised exploration of the data showed that the chromatin landscape of MM is widely altered as compared to NPCs. A supervised analysis of chromatin states revealed that MM globally shows a more active chromatin structure than NPCs. From over 40,000 regions identified with differential chromatin structure between MM and NPCs, 88% were de novo activated in neoplastic plasma cells. Analyzing the chromatin of individual genes, we observed that there were roughly ten times more genes gaining activity upon neoplastic transformation than those acquiring repressed chromatin marks. Interestingly, the genes showing more activate chromatin were enriched with biosynthesis and metabolic processes, while genes with repressed chromatin were related to gene ontology terms related to B cell signaling. Among those genes gaining de novo activity in MM, we selected several candidates and we are currently performing functional in vitro assays to explore their implication in MM pathogenesis. Furthermore, as extensive chromatin activation is a hallmark of MM, we are currently analyzing additional 15 MM cases and NPCs by ChIP-seq for H3K27ac (marking active promoters and enhancers) and ATAC-seq (marking active regulatory regions) to validate our initial findings and explore chromatin heterogeneity in MM.Collectively, our initial exploration of histone modification profiles in MM has revealed that MM cells acquire a more active chromatin landscape, with thousands of regions gaining activation as compared to NPCs. Reversing this global activation by epigenetic drugs, such as BET inhibitors, may represent an attractive therapeutic option for MM.During the meeting, updated information will be presented, including data from all 19 MM patients studied as well as functional data from new candidate genes involved in MM pathogenesis. DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Nucleolar organizer regions: genomic 'dark matter' requiring illumination.

Nucleoli form around tandem arrays of a ribosomal gene repeat, termed nucleolar organizer regions (NORs). During metaphase, active NORs adopt a characteristic undercondensed morphology. Recent evidence indicates that the HMG-box-containing DNA-binding protein UBF (upstream binding factor) is directly responsible for this morphology and provides a mitotic bookmark to ensure rapid nucleolar formation beginning in telophase in human cells. This is likely to be a widely employed strategy, as UBF is present throughout metazoans. In higher eukaryotes, NORs are typically located within regions of chromosomes that form perinucleolar heterochromatin during interphase. Typically, the genomic architecture of NORs and the chromosomal regions within which they lie is very poorly described, yet recent evidence points to a role for context in their function. In Arabidopsis, NOR silencing appears to be controlled by sequences outside the rDNA (ribosomal DNA) array. Translocations reveal a role for context in the expression of the NOR on the X chromosome in Drosophila Recent work has begun on characterizing the genomic architecture of human NORs. A role for distal sequences located in perinucleolar heterochromatin has been inferred, as they exhibit a complex transcriptionally active chromatin structure. Links between rDNA genomic stability and aging in Saccharomyces cerevisiae are now well established, and indications are emerging that this is important in aging and replicative senescence in higher eukaryotes. This, combined with the fact that rDNA arrays are recombinational hot spots in cancer cells, has focused attention on DNA damage responses in NORs. The introduction of DNA double-strand breaks into rDNA arrays leads to a dramatic reorganization of nucleolar structure. Damaged rDNA repeats move from the nucleolar interior to form caps at the nucleolar periphery, presumably to facilitate repair, suggesting that the chromosomal context of human NORs contributes to their genomic stability. The inclusion of NORs and their surrounding chromosomal environments in future genome drafts now becomes a priority.

Painting a Clearer Picture of Chromatin

Elucidating chromatin's 3D shape is critical to understanding its function, but the fine structure of chromatin domains remains poorly resolved. In a recent report in Nature, Boettiger etal. (2016) visualize chromatin in super-resolution, gaining unprecedented insight into chromatin architecture.

Analysis of PBase Binding Profile Indicates an Insertion Target Selection Mechanism Dependent on TTAA, But Not Transcriptional Activity.

Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome. We have shown that PBase binding mainly depends on the distribution of the tetranucleotide TTAA, which is not affected by the presence of PB DNA. Furthermore, PBase binding is negatively influenced by the methylation of CG sites in the genome. Analysis of a large collection of PB insertions in mice has revealed an insertion profile similar to the PBase binding profile. Interestingly, this profile is not correlated with transcriptional active genes in the genome or transcriptionally active regions within a transcriptional unit. This differs from what has been previously shown for P-element and retroviruses insertions. Our study provides an explanation for PB's genome-wide insertion distribution and also suggests that PB target selection relies on a new mechanism independent of active transcription and open chromatin structure.

The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation.

Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

Division of labor between IRF1 and IRF2 in regulating different stages of transcriptional activation in cellular antiviral activities.

BackgroundCellular antiviral activities are critically controlled by transcriptional activation of interferon-inducible genes, involving interferon regulatory factors (IRFs). Previous data suggested that IRF1 is an activator and IRF2 is a repressor, which functionally antagonize each other in transcriptional regulation. However, it is not clear how these two factors function to regulate cellular antiviral activities.ResultsWe show that IRF2 is critically required for the induction of the TLR3 and other interferon-inducible genes in a chromatin environment. While both IRF1 and IRF2 directly interact with the BAF chromatin remodeling complex, IRF2 is associated with the TLR3 promoter in the unstimulated state and IRF1 binding to the promoter is strongly induced by stimulation with interferon, suggesting that these two factors may function at different stages of gene induction in the recruitment of the BAF complex. IRF2 acts to maintain the basal level expression, an open chromatin structure, and active histone modification marks (H3K9, K14 acetylation and H3K4 tri-methylation) of the TLR3 promoter in the unstimulated state, while IRF1 serves to rapidly activate the promoter upon stimulation.ConclusionsIRF1 and IRF2 of the IRF family of transcription factors play distinct roles in cellular response to viral infection. IRF2 binds to TLR3 and other IFN-inducible gene promoters and maintains an active chromatin structure in the unstimulated state, which is required for their induction, while IRF1 binding to these promoters activates their transcription upon viral infection. Thus, the division of labor between the IRF transcription factor family members plays a pivotal role in coordinating the transcriptional activation in the cellular antiviral response.

KLF1 stabilizes GATA-1 and TAL1 occupancy in the human β-globin locus

KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the β-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the β-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the β-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the β-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping.

SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.

Chromatin profiling reveals regulatory network shifts and a protective role for hepatocyte nuclear factor 4α during colitis.

Transcriptional regulatory mechanisms likely contribute to the etiology of inflammatory bowel disease (IBD), as genetic variants associated with the disease are disproportionately found at regulatory elements. However, the transcription factors regulating colonic inflammation are unclear. To identify these transcription factors, we mapped epigenomic changes in the colonic epithelium upon inflammation. Epigenetic marks at transcriptional regulatory elements responded dynamically to inflammation and indicated a shift in epithelial transcriptional factor networks. Active enhancer chromatin structure at regulatory regions bound by the transcription factor hepatocyte nuclear factor 4α (HNF4A) was reduced during colitis. In agreement, upon an inflammatory stimulus, HNF4A was downregulated and showed a reduced ability to bind chromatin. Genetic variants that confer a predisposition to IBD map to HNF4A binding sites in the human colon cell line CaCo2, suggesting impaired HNF4A binding could underlie genetic susceptibility to IBD. Despite reduced HNF4A binding during inflammation, a temporal knockout model revealed HNF4A still actively protects against inflammatory phenotypes and promotes immune regulatory gene expression in the inflamed colonic epithelium. These findings highlight the potential for HNF4A agonists as IBD therapeutics.

Establishment of active chromatin structure at enhancer elements by mixed-lineage leukemia 1 to initiate estrogen-dependent gene expression

A number of genome-wide analyses have revealed that estrogen receptor α binding to and regulation of its target genes correlate with binding of FOXA1, a pioneer factor, to nearby DNA sites in MCF-7 breast cancer cells. The enhancer element-specific histone H3K4me1/2 mark is enriched at the specific FOXA1/ERα recruitment sites in chromatin, but the mechanism by which these enhancer marks are established in chromatin before hormone treatment is unclear. Here, we show that mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERα to the enhancer element. Our study defines the mechanism by which MLL1 nucleates histone H3K4 methylation marks in CpG-enriched regions to maintain permissive chromatin architecture and allow FOXA1 and estrogen receptor α binding to transcriptional regulatory sites in breast cancer cells.

A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain

Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult αD globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult αD gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the αD adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure.

Prior epigenetic priming of cytokine genes in naive T cells is required for their subsequent activation by inducible enhancers

The inducible lL-3 and GM-CSF genes can only be efficiently expressed in T cells once they have been through a previous cycle of activation and undergone a process termed blast cell transformation [1]. An initial stimulus is required to bring naive T cells out of the resting state and into the cell cycle. This process is triggered by T cell receptor (TCR) stimulation, takes about 24 hours, and is associated with extensive nuclear remodeling. Once primed by a cycle of activation, T blast cells can maintain their ability to express lL-3 and GM-CSF for many cell divisions without the continual need for additional stimuli. In contrast, the lL-3 and GM-CSF genes cannot be induced in naive T cells that have never received a TCR stimulus. We show that this pattern of regulation of the IL-3/GM-CSF locus is controlled at two distinct levels: (1) During blast cell transformation, the IL-3/GM-CSF locus acquires an extensive array of DNase I Hypersensitive Sites (DHSs) which are then maintained indefinitely for many cell cycles [1,2]. These primed DHSs are marked by me2K4 histone H3, and they also persist in non-dividing memory T cells in the peripheral blood [1]. These DHSs are absent in the thymus, spleen T cells, and naive T cells in the blood. These DHSs do not function as classical enhancers, and we propose that they serve to maintain an active chromatin structure in previously activated T cells. (2) The expression of the lL-3 and GM-CSF genes is in each case dependent on the activation of inducible upstream enhancers. The lL-3 and GM-CSF enhancers appear as inducible DHSs T blast cells within 20 min of stimulation, and only acquire the me2K4H3 modification after stimulation. These enhancers are dependant on the TCR inducible factors NFAT and AP-1. However, although AP-1 and NFAT family mRNAs are efficiently expressed in both naive T cells and in T blast cells, the enhancers only respond to induction of these factors in T blast cells. The inducible DHSs remain completely undetectable in naive T cells even after 4 hours of stimulation with direct activators of TCR signaling pathways (PMA and Calcium ionophore). We propose that T blast cells and memory T cells have developed a strategy of maintaining a discrete class of DHS as epigenetic memory modules that support an accessible chromatin environment and thereby prime inducible genes for efficient re-activation when memory cells re-encounter Ag stimuli. In the absence of the priming elements, the lL-3/GM-CSF locus appears to remain inaccessible to transcription factors that are otherwise very efficient at recruiting the chromatin remodelers that create DHSs within the inducible enhancers.

Nuclear Repositioning Precedes Promoter Accessibility and Is Linked to the Switching Frequency of a Plasmodium falciparum Invasion Gene

Variation of surface adhesins, such as the Plasmodium falciparum erythrocyte invasion ligand PfRh4, is critical for virulence and immune evasion in many microbes. While phenotypic switching is linked to transcriptional changes and chromatin function, the determinants of switching frequency remain poorly defined. By expressing a prokaryotic DNA methylase in P.falciparum, we directly assayed accessibility of transcriptionally active and silent chromatin at the PfRh4 locus. Parasites selected for invivo PfRh4 activation show a reversible increase in promoter accessibility and exhibit perinuclear repositioning of the locus from a silent to a conserved activation domain. Forced activation of a proximal gene results in a similar repositioning of the PfRh4 locus, witha concomitant increase in PfRh4 activation ina subpopulation of parasites and promoter accessibility correlating with actively transcribed loci. Thus, nuclear repositioning is associated with increased P.falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

Aberrant Chromatin Modifications Mediate Ectopic Expression of Homeobox Gene NKX2-1 in B-Cell Lymphoma

Abstract 3494Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, and when deregulated promote cell transformation in multiple cancers including hematopoietic malignancies. In this context several members of the NKL family of homeobox genes are aberrantly expressed in acute T-cell leukemia by chromosomal aberrations. Here, analysis of 20 cell lines of T- and B-cell leukemia/lymphoma by expression arrays (Affymetrix, HGU133plus2) revealed exclusive activity of NKL homeobox gene NKX2-1 in a diffuse large B-cell lymphoma (DLBCL) cell line. NKX2-1 is physiologically expressed in embryonic lung and thyroid tissues where it regulates differentiation. RQ-PCR analysis of gene expression in primary hematopoietic samples, including bone marrow, lymph node, thymus, peripheral mononuclear blood cells, T-cells and B-cells, confirmed silencing therein highlighting ectopic expression of NKX2-1 in the cell line. Copy number analysis by genomic array data (Broad Institute), spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) excluded chromosomal rearrangements at the NKX2-1 locus in expressing cells. Comparative expression analysis of NKX2-1 negative DLBCL cell lines implicated several candidate genes involved in NKX2-1 regulation, variously encoding transcription factors (TFs), chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed TF HEY1 in ectopic NKX2-1 expression and NKX2-1 in HEY1 expression in DLBCL cells, indicating reciprocal activation of these TFs. Moreover, chromatin immunoprecipitation (ChIP) analysis demonstrated direct binding of NKX2-1 to the HEY1 promoter. HEY1 belongs to the basic helix-loop-helix family disturbing lymphoid differentiation if deregulated. Enhanced expression levels of histone H3K4 methyltransferase MLL correlated with downstream rearrangement and amplification of the MLL-locus at 11q23. SiRNA-mediated knockdown of MLL was accompanied by reduced expression of NKX2-1 but not of HEY1, showing that MLL supports expression of NKX2-1. Furthermore, ChIP analyses demonstrated presence of both activatory H3K4me3 and inhibitory H3K27me3 at the promoter region of NKX2-1, while at the HEY1 promoter only H3K27me3 was detected. Such bivalent histone marks have been described for developmental genes in progenitor cells, indicating a permissive role for aberrant chromatin structures at the NKX2-1 locus in this cell line. Chromosomal alteration del(6p22) as detected by SKY and subsequently mapped by FISH was shown to target the histone gene locus HIST1. Expression analysis at the RNA and protein levels showed elevated expression of core-histones including H2B. Additionally, mono-ubiquitinated H2B was strongly enhanced in this DLBCL cell line when analyzed by Western blot. This histone mark supports the MLL-mediated formation of active chromatin structures, suggesting cooperative action of the chromosomal aberrations targeting MLL and HIST1. Array data also indicated aberrant expression of polycomb repressor complex 2 (PRC2) members which counteract the activity of MLL. Accordingly, siRNA-mediated knockdown analyses demonstrated regulatory impacts of HOPX, E2F6 and JMJD3 in NKX2-1 expression. The potential impact of signaling pathways in NKX2-1 expression, comprising NFkB, SMADs and phosphodiesterases was confirmed by treatments with TNFa, TGFb and cAMP/cGMP, respectively. Taken together, we have identified ectopic expression of NKX2-1 in DLBCL cells, involved in an oncogenic regulative network which may compromise B-cell differentiation via activation of HEY1. Combined analyses of chromosomal alterations and comparative gene expressions identified aberrant chromatin structures underlying expression of NKX2-1, representing the central player in that network. Therefore, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancy. Disclosures:No relevant conflicts of interest to declare.