Helmut Hofemeister

The Transcriptional and Epigenomic Foundations of Ground State Pluripotency

Summary Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as “2i” postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, “We thank I. Cheesman for helpful discussions.” The error has been corrected for all versions of the article.

Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant

Trimethylation of histone H3 lysine 4 (H3K4me3) at the promoters of actively transcribed genes is a universal epigenetic mark and a key product of Trithorax group action. Here, we show that Mll2, one of the six Set1/Trithorax-type H3K4 methyltransferases in mammals, is required for trimethylation of bivalent promoters in mouse embryonic stem cells. Mll2 is bound to bivalent promoters but also to most active promoters, which do not require Mll2 for H3K4me3 or mRNA expression. By contrast, the Set1 complex (Set1C) subunit Cxxc1 is primarily bound to active but not bivalent promoters. This indicates that bivalent promoters rely on Mll2 for H3K4me3 whereas active promoters have more than one bound H3K4 methyltransferase, including Set1C. Removal of Mll1, sister to Mll2, had almost no effect on any promoter unless Mll2 was also removed, indicating functional backup between these enzymes. Except for a subset, loss of H3K4me3 on bivalent promoters did not prevent responsiveness to retinoic acid, thereby arguing against a priming model for bivalency. In contrast, we propose that Mll2 is the pioneer trimethyltransferase for promoter definition in the naïve epigenome and that Polycomb group action on bivalent promoters blocks the premature establishment of active, Set1C-bound, promoters.

Recombineering BAC transgenes for protein tagging.

Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.

Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.

Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation).

The histone methyltransferase KMT2B is required for RNA polymerase II association and protection from DNA methylation at the MagohB CpG island promoter.

ABSTRACT KMT2B (MLL2/WBP7) is a member of the MLL subfamily of H3K4-specific histone lysine methyltransferases (KMT2) and is vital for normal embryonic development in the mouse. To gain insight into the molecular mechanism underlying KMT2B function, we focused on MagohB, which is controlled by a CpG island promoter. We show that in cells lacking Mll2—the gene encoding KMT2B—the MagohB promoter resides in inaccessible chromatin and is methylated. To dissect the molecular events leading to the establishment of silencing, we performed kinetic studies in Mll2-conditional-knockout embryonic stem cells. KMT2B depletion was followed by the loss of the active chromatin marks and progressive loss of RNA polymerase II binding with a concomitant downregulation of MagohB expression. Once the active chromatin marks were lost, the MagohB promoter was rapidly methylated. We demonstrate that in the presence of KMT2B, neither transcription elongation nor RNA polymerase II binding is required to maintain H3K4 trimethylation at the MagohB promoter and protect it from DNA methylation. Reexpression of KMT2B was sufficient to reinstate an active MagohB promoter. Our study provides a paradigm for the idea that KMT2 proteins are crucial components for establishing and maintaining the transcriptionally active and unmethylated state of CpG island promoters.

Erratum: Corrigendum: BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals

Ina Poser, Mihail Sarov, James R A Hutchins, Jean-Karim Hériché, Yusuke Toyoda, Andrei Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P White, Steffen Dietzel, Karl Mechtler, Richard Durbin, A Francis Stewart, Jan-Michael Peters, Frank Buchholz & Anthony A Hyman Nat. Methods 5, 409–415 (2008).

Erratum: BAC TransgeneOmics: A high-throughput method for exploration of protein function in mammals (Nature Methods (2008) vol. 5 (409-415))

Ina Poser, Mihail Sarov, James R A Hutchins, Jean-Karim Hériché, Yusuke Toyoda, Andrei Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P White, Steffen Dietzel, Karl Mechtler, Richard Durbin, A Francis Stewart, Jan-Michael Peters, Frank Buchholz & Anthony A Hyman Nat. Methods 5, 409–415 (2008).