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

ERG Controls B Cell Development by Promoting <i>Igh </i>V-to-DJ Recombination

B cell development depends on the coordinated expression and cooperation of several transcription factors. Here we show that the transcription factor ETS-related gene (ERG) is crucial for normal B cell development and that its deletion results in a substantial loss of bone marrow B cell progenitors and peripheral B cells, as well as a skewing of splenic B cell populations. We find that ERG-deficient B lineage cells exhibit an early developmental block at the pre-B cell stage and proliferate less. The cells fail to express the immunoglobulin heavy chain due to inefficient V-to-DJ recombination, and cells that undergo recombination display a strong bias against incorporation of distal V gene segments. Furthermore, antisense transcription at PAX5-activated intergenic repeat (PAIR) elements, located in the distal region of the Igh locus, depends on ERG. These findings show that ERG serves as a critical regulator of B cell development by ensuring efficient and balanced V-to-DJ recombination

Retroviral Insertional Mutagenesis Screen in a C/EBPalpha Proliferative Genetic Background.

Abstract The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours after birth. In contrast, homozygous knockin mice in which the wild type Cebpa allele has been replaced with a mutant allele (BRM2) deficient in repressing the activity of E2F family members, are viable. At 8 weeks of age these animals display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older BRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia. These findings strongly suggest that secondary mutations in other loci must occur during the phenotypic progression. In order to identify genes that cooperate with C/EBPalpha in the development of leukemia in BRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn BRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus and when disease is evident the mice are euthanized and analyzed. As expected the BRM2/BRM2 mice have a shorter latency than wildtype mice (182 vs. 260 days). The mice have enlarged spleen, thymus, and lymph nodes and were further characterized by histology, flow cytometry and Southern blotting in order to determine the hematopoietic phenotypes. Most abundantly was the AML-like phenotype, but also T-cell lymphomas are developing. Finally, the loci carrying retroviral insertions loci are identified through a splinkerette-aided PCR strategy. This study provides a better understanding of the genes involved in the development of myeloid leukemia.</jats:p

Integrative analysis of histone ChIP-seq and transcription data using Bayesian mixture models

Motivation: Histone modifications are a key epigenetic mechanism to activate or repress the transcription of genes. Datasets of matched transcription data and histone modification data obtained by ChIP-seq exist, but methods for integrative analysis of both data types are still rare. Here, we present a novel bioinformatics approach to detect genes that show different transcript abundances between two condi-tions putatively caused by alterations in histone modification. Results:We introduce a correlation measure for integrative analysis of ChIP-seq and gene transcription data measured by RNA sequencing or microarrays and demonstrate that a proper normalization of ChIP-seq data is crucial. We suggest applying Bayesian mixture models of different types of distributions to further study the distribution of the correlation measure. The implicit classification of the mixture models is used to detect genes with differences between two conditions in both gene transcription and histone modification. The method is applied to different datasets, and its superiority to a naive separate analysis of both data types is demonstrated. Availability and implementation: R/Bioconductor package epigenomix. Contact

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

BackgroundTranscription 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 findingsHere, 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/significanceTaken 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

A fraction of the one-year old <i>Cebpa</i>^S248A/S248A mice develops a myeloid disorder with biased lineage choice.

<p>(A) Flow cytometry analysis of the myeloerythroid progenitor compartment in BMs from <i>Cebpa</i>^KI/KI (n = 17) and <i>Cebpa</i>^S248A/S248A (n = 23) mice. (B) Seven out of 23 <i>Cebpa</i>^S248A/S248A mice (termed “progressed”) had a skewed lineage distribution with a decreased GMP/preMegE ratio compared to <i>Cebpa</i>^KI/KI. Black line indicates cut-off. Cut-off was defined as mean of <i>Cebpa</i>^KI/KI−standard deviation. (C) Quantification of the data from (A). Numbers of mice in each of the groups were as follows: <i>Cebpa</i>^KI/KI (n = 17), <i>Cebpa</i>^S248A/S248A (n = 16) and progressed <i>Cebpa</i>^S248A/S248A (n = 7) 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

Distribution of mice based on GMP/preMegE ratio and LSK numbers.

<p>(A) Mice (age 1–2 years old) were binned based on their GMP to preMegE progenitor ratio (Cebpa^KI/KI−standard deviation; mean of <i>Cebpa</i>^KI/KI+/−standard deviation and >mean of <i>Cebpa</i>^KI/KI+standard deviation), (P = 0,012, Fishers exact test). (B) Mice (age 1–2 years old) were binned based on their level of LSK cells (Cebpa^KI/KI−standard deviation; mean of <i>Cebpa</i>^KI/KI+/−standard deviation and >mean of <i>Cebpa</i>^KI/KI+standard deviation), (P = 0,14, Fishers exact test). Total number of 1–2 year-old mice: <i>Cebpa</i>^KI/KI (n = 25), <i>Cebpa</i>^S248A/S248A (n = 44). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038841#pone-0038841-t001" target="_blank">Table 1</a> for an overview of the phenotypes.</p

C/EBPα-S248A cannot induce differentiation of 32Dcl3 cells.

<p>(A) Western blot analysis of 32Dcl3 clones expressing C/EBPα-ER (wt) or C/EBPα-S248A-ER (S248A). (B) Flow cytometry analysis of expression of the granulocytic-monocytic markers Mac-1 and Gr-1 following 4-OHT addition to the C/EBPα-ER or C/EBPα-S248A-ER expressing clones. (C) Quantification of the flow cytometry data in (B) from two experiments using two independent wt-ER and S248A-ER clones (mean +/− standard deviation). (D) Quantification of the G1/S ratios determined by flow cytometry of BrdU and Propidium Iodide stained cell cultures. The data is from two experiments (mean +/− standard deviation).</p