John Arne Dahl

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

A rapid micro chromatin immunoprecipitation assay (ChIP)

Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin immunoprecipitation (ChIP) is a robust technique for studying protein–DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, or involve lengthy procedures. We describe here a 1-day micro ChIP (μChIP) protocol suitable for up to eight parallel histone and/or transcription factor immunoprecipitations from a single batch of 1,000 cells. μChIP technique is also suitable for monitoring the association of one protein with multiple genomic sites in 100 cells. Alterations in cross-linking and chromatin preparation steps also make μChIP applicable to ∼1-mm3 fresh- or frozen-tissue biopsies. From cell fixation to PCR-ready DNA, the procedure takes ∼8 h for 16 ChIPs.

High‐resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence

The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell‐derived cultures of human adipose stem cells to senescence. G‐banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high‐resolution microarray‐based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long‐term (∼6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene‐rich telomeric and sub‐telomeric regions on three chromosomes in early passage. This however, was detected only in a sub‐population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long‐term ASC cultures, at least under the culture conditions used here.

Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells.

Chromatin immunoprecipitation (ChIP) is a key technique for studying protein‐DNA interactions and mapping epigenetic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q2)ChIP assay suitable for histone and transcription factor immunoprecipitation from chromatin amounts equivalent to as few as 100 cells. DNA‐protein cross‐linking in suspension in presence of butyrate, elimination of background chromatin through a tube shift after washes, and a combination of cross‐link reversal, protein digestion, increased antibody‐bead to chromatin ratio, and DNA elution into a single step considerably improve ChIP efficiency and shorten the procedure. We used Q2ChIP to monitor changes in histone H3 modifications on the 5′ regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic‐acid‐mediated human embryonal carcinoma cell differentiation. Quantitative polymerase chain reaction analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation. Di‐ and trimethylation of H3K4 remain relatively unaltered. In contrast, PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. PAX6 undergoes H3K9 acetylation and enhanced H3K4 trimethylation upon transcriptional activation. Q2ChIP of the transcription factor Oct4 demonstrates its dissociation from the NANOG promoter upon differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences. The results demonstrate ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.

Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation.

Our understanding of the significance of interactions of proteins with DNA in the context of gene expression, cell differentiation or to some extent disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP has been widely used to map the localization of post-translationally modified histones or histone variants on the genome or on a specific gene locus, or to map the association of transcription factors or chromatin modifying enzymes to the genome. In spite of its power, ChIP is a cumbersome procedure and typically requires large numbers of cells. This review outlines variations elaborated on the ChIP assay to shorten the procedure, make it suitable for small cell numbers and unravel the multiplicity of histone modifications on a single locus. In addition, the combination of ChIP assays with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modifications and transcription factor occupancy sites throughout the genome and in a high-resolution manner throughout a genomic region of interest. We also review applications of ChIP to the mapping of histone modifications or transcription factor binding at the genome-wide level. Finally, we speculate on future perspectives opened by the combination of emerging ChIP-related technologies.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (μChIP–seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the β-glucosyltransferase from T4 bacteriophage creating β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC). The β-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level.

Histone H3 Lysine 27 Methylation Asymmetry on Developmentally-Regulated Promoters Distinguish the First Two Lineages in Mouse Preimplantation Embryos

First lineage specification in the mammalian embryo leads to formation of the inner cell mass (ICM) and trophectoderm (TE), which respectively give rise to embryonic and extraembryonic tissues. We show here that this first differentiation event is accompanied by asymmetric distribution of trimethylated histone H3 lysine 27 (H3K27me3) on promoters of signaling and developmentally-regulated genes in the mouse ICM and TE. A genome-wide survey of promoter occupancy by H3K4me3 and H3K27me3 indicates that both compartments harbor promoters enriched in either modification, and promoters co-enriched in trimethylated H3K4 and H3K27 linked to developmental and signaling functions. The majority of H3K4/K27me3 co-enriched promoters are distinct between the two lineages, primarily due to differences in the distribution of H3K27me3. Derivation of embryonic stem cells leads to significant losses and gains of H3K4/K27me3 co-enriched promoters relative to the ICM, with distinct contributions of (de)methylation events on K4 and K27. Our results show histone trimethylation asymmetry on promoters in the first two developmental lineages, and highlight an epigenetic skewing associated with embryonic stem cell derivation.

μChIP—a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies

Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein–DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no protocols for fast and parallel ChIPs of post-translationally modified histones from small cell numbers or biopsies, and importantly, no protocol allowing for investigations of transcription factor binding in small cell numbers. We report here the development of a micro (μ) ChIP assay suitable for up to nine parallel quantitative ChIPs of modified histones or RNA polymerase II from a single batch of 1000 cells. μChIP can also be downscaled to monitor the association of one protein with multiple genomic sites in as few as 100 cells. μChIP is applicable to small fresh tissue biopsies, and a cross-link-while-thawing procedure makes the assay suitable for frozen biopsies. Using μChIP, we characterize transcriptionally permissive and repressive histone H3 modifications on developmentally regulated promoters in human embryonal carcinoma cells and in osteosarcoma biopsies. μChIP creates possibilities for multiple parallel and rapid transcription factor binding and epigenetic analyses of rare cell and tissue samples.

Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads

We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.