Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated DNA, making them prone to introduce experimental biases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-014-1195-4) contains supplementary material, which is available to authorized users

Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML

The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human mutant AML and the corresponding mouse model, we identified , encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene. Increased expression of CD73, mediated by the CEBPA-p30 isoform, sustained leukemic growth via the CD73/A2AR axis. Notably, targeting of this pathway enhanced survival of AML-transplanted mice. Our data thus indicate a first-in-class link between a cancer driver mutation in a TF and a druggable, direct transcriptional target

Phosphorylation of Serine 248 of C/EBPα Is Dispensable for Myelopoiesis but Its Disruption Leads to a Low Penetrant Myeloid Disorder with Long Latency

BACKGROUND: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. CONCLUSIONS/SIGNIFICANCE: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context

Nutrient Availability as a Mechanism for Selection of Antibiotic Tolerant Pseudomonas aeruginosa within the CF Airway

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues

The phenotype in <i>Cebpa</i>^S248A/S248A is cell-intrinsic.

<p>(A) Flow cytomtery analysis of recipients transplanted with BM from <i>Cebpa</i>^KI/KI (n = 7) and <i>Cebpa</i>^S248A/S248A (n = 7) mice. (B) Quantification of data from (A). Four out of seven of the <i>Cebpa</i>^S248A/S248A transplanted mice displayed a reduced GMP to preMegE ratio (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038841#pone.0038841.s005" target="_blank">Figure S5</a>) and was termed progressed. Numbers of mice in the other groups of transplanted mice were as follows: <i>Cebpa</i>^KI/KI (n = 7) and <i>Cebpa</i>^S248A/S248A (n = 3) mice. P values designate significance between progressed <i>Cebpa</i>^S248A/S248A and <i>Cebpa</i>^KI/KI, ns = not significant (mean +/− standard deviation).</p

<i>Cebpa</i>^S248A/S248A BM cells have a competitive advantage in comparison to <i>Cebpa</i>^KI/KI BM cells.

<p>(A) Whole BM from <i>Cebpa</i>^KI/KI and <i>Cebpa</i>^S248A/S248A mice (CD45.2) was mixed in a 1∶1 ratio with whole competitor BM (CD45.1) and transplanted into lethally irradiated mice. BM donor contribution was assessed 18 weeks post-transplantation. <i>Cebpa</i>^KI/KI (n = 7) and <i>Cebpa</i>^S248A/S248A (n = 6) BM cells. (B) Input BM for the secondary transplantation show similar levels of <i>Cebpa</i>^S248A/S248A and <i>Cebpa</i>^KI/KI contribution in the two input samples. (C, D) BM donor contribution in whole BM (wBM) or in the LSK compartment of the secondary recipients was analyzed after 21 (C) and 34 (D) weeks. <i>Cebpa</i>^S248A/S248A transplanted mice (n = 4; black bars); <i>Cebpa</i>^KI/KI transplanted mice (n = 4; white bars).</p

Young <i>Cebpa</i>^S248A/S248A mice show no abnormalities in the hematopoietic stem and progenitor compartment.

<p>(A) Flow cytometry analysis of the stem and progenitor compartment in the BM from <i>Cebpa</i>^KI/KI and <i>Cebpa</i>^S248A/S248A. (B) Quantification of the data in (A). There were no significant changes in <i>Cebpa</i>^KI/KI (n = 6) and <i>Cebpa</i>^S248A/S248A (n = 6) mice (mean +/− standard deviation). (C) Colony forming unit assays of BM cells from <i>Cebpa</i>^KI/KI (n = 4) and <i>Cebpa</i>^S248A/S248A (n = 4) mice (mean +/− standard deviation). (D) Serial replating of the primary colonies from (C) showing the behavior of 3 individual mice of each genotype.</p