Effect of Gfi1 36N variant on genome-wide H3K9 Acetylation patterns
Ontology highlight
ABSTRACT: ChIP-Seq Analysis of H3K9Ac in pairs of mouse and human samples carrying either the Gfi136S or the GFi136N variants. The objective of the study was to identify the changes in H3K9 acetylation at gene promoters that occur in samples expressing the 36N variant of the Gfi1 gene. 3 pairs of bone-marrow AML samples were obtained from mice where 1 mouse in each pair was homozygous for Gfi136S and 1 heterozygous for Gfi136N, or homozygous for 36N in one case. 2 pairs of AML samples were obtained from human patients were 1 patient was homozygous for Gfi1 36S and one was heterozygous for Gfi1 36N. H3 and H3K9Ac ChIP-Seq was carried out on each sample.
Project description:We investigated the RNAPII and γH2AX occupancy genome wide by ChIP-Seq in MLL2 F/F and FC/FC80 MEF cells. We found that a week after MLL2 excision (FC/FC cells), a group of genes present higher levels of γH2AX and RNAPII near the TSS, as compared to the control (F/F cells). H3K4Me1, H3K4M2 and H3K4Me3 levels near the TSS were also studied. There is a total of 52 samples. 3 independent replicates for each experiment were performed. H3, H2AX and IgG ChIPs were used for normalisation or as controls.The experiments were performed using immortalised mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).
Project description:We investigated the genomewide binding pattern of prevalent p53 gain-of-function (GOF) mutants by ChIP-seq, in a panel of breast cancer cell lines. We assessed the genomewide changes of H3K4me3 upon GOF p53 knockdown in MDA-MB-468 breast cancer cells bearing the p53 R273H mutation. This study uses ChIP-seq of H3K4me3 and histone H3 in wild-type or p53 R172H knock-in MEFs. Additionally, this study examines the transcriptome of wild-type or p53 R172H knock-in MEFs using polyA+ RNA-seq.
Project description:The plant circadian clock exerts a critical role in the regulation of multiple biological processes including responses to biotic and abiotic stresses. It is estimated that the clock regulates up to 80% of the transcriptome in Arabidopsis, thus understanding the molecular mechanisms that control this rhythmic transcriptome requires identification of the targets of each clock component. The Arabidopsis core clock is partially comprised of a transcriptional regulatory loop between the MYB domain containing transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION1 (TOC1). As a key component of the clock, CCA1 is able to initiate and set the phase of clock-controlled rhythms. CCA1 regulates the transcription of several genes by directly binding to the evening element (EE) motif primarily found in the promoters of evening expressed genes. Using a genome-wide approach we have identified direct targets of CCA1 in plants grown in constant (LL) and driven conditions (LD). These CCA1 targets are enriched for a myriad of biological processes and stress responses. While many of these target genes are evening phased and contain the EE in their promoter regions, a significant subset is morning phased and lack an EE. Furthermore, several CCA1 targets do not cycle in either LL or LD or both. Expression analysis in CCA1 overexpressing plants confirms CCA1 regulation of analyzed targets. Our results emphasize an expanded role for the circadian clock in regulation of key pathways in Arabidopsis, and provide a comprehensive and solid resource for future functional studies. ChIP-Seq of CCA1-GFP plants under control of the CCA1 promoter in continuous light and diel conditions
Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, Krüppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role. ChIP-Seq experiments were performed on four HEK293 cell lines, two each stably expressing AZF or KLF3FD.AZF or KLF3FD only (two biological replicates). Input samples were included as controls.
Project description:We previously demonstrated by genomic and bioinformatical approaches that human macrophage (MΦ) activation is best described by a spectrum model (Xue et al, Immunity, 2014). MΦ integrate exogenous input signals on transcriptional level in a unique fashion to generate specific functional programs, enabling the plasticity in disease-related pathophysiologies. Such versatile responsiveness requires fast changes of transcription mediated by transcriptional regulators (TRs) or epigenomic changes. To better understand the principles of this regulation during human MΦ activation, we assessed histone modifications including H3K4me1, H3K4me3, H3K27me3, and H3K27Ac by ChIP-sequencing allowing us to characterize the functional state of promoters (active, poised, repressed) and enhancers (active, inactive, intermediate). Using transcriptome data from our MΦ spectrum model, we generated a co-regulation network of all TRs. Next, we overlaid epigenomic information and transcriptional changes of major TRs over time onto the TR network. We observed that input signals like IFNγ or TNFα induce a specific network of TRs that are transcriptionally regulated themselves, the combination of regulated TRs changes over time with a boost of transcriptional regulation of dozens of TRs 4 to 12 hrs post input signal exposure, almost all TRs within the network show active promoters, even if the TR itself is not expressed, and similar results are obtained for enhancers with open or at least intermediated states. These findings strongly suggest that in MΦ, the TR-defined cellular â??switch panelâ?? is always accessible thereby allowing MΦ to quickly respond to the diverse input signal repertoire from the environment. Epigenetic analysis of promoter and enhancer sites in primary human macrophage subtypes and correlation to RNA-seq expression data
Project description:Transcriptional repressor Growth factor independence 1 (GFI1) is a key regulator of haematopoiesis. We previously established that the germline variant GFI1-36N promotes acute myeloid leukemia (AML) development, however the mechanism is not full elucidated. Here using multi-omics approach, we show GFI1-36N expression impedes DNA repair in leukemic cells. We demonstrate the presence of GFI1-36N is associated with increased frequency of chromosomal aberrations and mutational burden in murine and human AML cells. In particular, GFI1-36N modulates DNA repair pathways, O6-methylguanine-DNA-methyltransferase (MGMT) and homologous recombination repair (HR). Mechanistically, GFI1-36N exhibits impaired binding to Ndrg1 promoter element compared to GFI1-36S (wild type), causing decreased NDRG1 levels consequently leading to suppression of MGMT expression, imprinted at the transcriptome and proteome, thus leaving the AML cells vulnerable to DNA damaging agents. Targeting MGMT via temozolomide and HR via olaparib caused specifically extensive lethality in in vitro and ex vivo human and AML samples expressing GFI1-36N. Whereas the effects were insignificant on non-malignant GFI1-36S or GFI1-36N cells. Further, mice transplanted with GFI1-36N leukemic cells treated with combination of temozolomide and olaparib had a significantly longer AML-free survival than mice transplanted with GFI1-36S leukemic cells. In summary, we show that GFI1-36N disturbs DNA repair activity via NDRG1-MGMT axis and thus provides critical insights into novel therapeutic option for AML presented with GFI1-36N variant. Key Points Presence of GFI1-36N impedes Homologous DNA and MGMT DNA repair selectively in AML cells via the NDRG1-MGMT axis. Use of temozolomide and olaparib allows selectively targeting GFI1-36N leukemic cells. Introduction Gfi1 is a transcription factor which regulates the development of haematopoietic cells as well as neuronal and intestinal epithelial cells 1-5. We reported that a variant of GFI1, denominated GFI1-36N (characterized by an exchange of serine to asparagine at position 36), has a prevalence of 5-7% in a healthy control population but is found at an increased frequency of 10-15% among MDS and AML patients 6,7. The expression of germline variant GFI1-36N predisposes the carriers to develop de novo AML and MDS and correlates with a poor prognosis 6,7. Recently, we and other showed that malignant cells with GFI1-36N variant have increased H3K9-acetylation at target genes resulting in higher expression of genes required for cell survival and proliferation 8. GFI1 exerts its repressive role by recruiting histone-modifying enzymes (deacetylases HDAC1-3, demethylase LSD1, methyl transferase G9a) and regulates the accessibility of DNA to its target genes such as Hoxa9, Pbx1, Meis1, CSF1 and CSFR1 9-15. We also showed that GFI1 regulates apoptosis through its regulation of p53 in lymphoblastic leukemia 16 and we have demonstrated that GFI1 facilitates DNA repair 17. However, it is not known how these activities are affected in the GFI1-36N variant and whether the ability of GFI1 to regulate DNA repair pathways is maintained and how this might affect the development of myeloid malignancies. In this study, we leveraged multi-omics profiling to gain mechanistic insights into the molecular architecture that drives leukemia in the presence of GFI1-36N. We provide evidence that GFI1-36N interferes with DNA in leukemic myeloid cells, which leads to a higher frequency of genetic aberrations in MDS/AML cells. We also show that GFI1-36N myeloid leukemic cells are more sensitive to targeting MGMT and HR repair deficient cells, which opens a new selective therapeutic window to treat AML/MDS.
Project description:Growth factor independent-1 (Gfi1) is a zinc finger transcription factor with a SNAG amino-terminal repressor domain and is critically required for normal myelopoesis. Gfi1 is normally expressed in HSCs and induced during differentiation from CMPs to GMPs. Gfi1 loss-of-function mutation in mice and humans induces an arrest during myeloid differentiation and an absolute block to the formation mature neutrophils. This comparative microarray study was initiated to identify microRNAs which are potentially regulated by Gfi1 during granulopoiesis. The Gfi1 null allele from Orkin (deletion of exons 2 and 3) was backcrossed to B6 or Balb/c mice for at least 7 generations. RNA was isolated from low density bone marrow samples from Gfi1+/+ and Gfi1-/- B6 littermates, as well as Gfi1+/+ and Gfi1-/- Balb/c littermates.
Project description:Understanding factors that drive development and function of the sinoatrial node (SAN) is crucial to development of potential therapies for sinus arrhythmias, including potential generation of biological pacemakers. Here, we identify a key cell autonomous role for the LIM homeodomain transcription factor ISL1 for survival, proliferation and function of pacemaker cells throughout development. Chromatin immunoprecipitation assays performed utilizing antibody to ISL1 in chromatin extracts from FACS purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes critical for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct downstream targets of ISL1 in SAN cells. Our studies represent the first in vivo ChIP-seq studies for SAN cells which provide a basis for further exploration of factors critical to SAN formation and function and highlight the potential for utilization of ISL1 in combination with other SAN transcription factors for generating pacemaker cells for therapy or drug screening purposes. ISL1 ChIP-seq profiling was performed in Hcn4-H2BGFP SAN cells purified from neonatal hearts.
Project description:The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer binding protein b (CEBPb), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator activated receptor g2 (PPARg2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is required for adipogenesis but need not be active in the mature adipocyte, transiently functions with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARg2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocytes involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process. Genomic occupancy profiled by high throughput sequencing (ChIP-seq) from 3T3-L1 cells during differentiation for H3K9Ac, CEBPb and GR.
Project description:This study investigates the proteomic alterations in bone marrow neutrophils isolated from 5-8 week old Gfi1+/-, Gfi1K403R/-, Gfi1R412X/-, and Gfi1R412X/R412X mice using the SWATH-MS technique. This dataset consists of 26 raw MS files, comprising 13 DDA (for spectral library generation)and 13 DIA (for SWATH) runs on a TripleTOF 5600 plus (SCIEX). Our findings revealed significant changes in the expression of neutrophil granule proteins and NADPH-oxidase complex components in Gfi1-mutant neutrophils.