Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes1. This includes acetylation of H3 on lysine 27 (H3K27ac), which blocks the deposition of polycomb mediated H3K27me32. H3K27ac is also widely used to identify active enhancers3,4, and the assumption has been that profiling of H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of H3 (H3K64ac and H3K122ac) marks active gene promoters and also a subset of active enhancers. Moreover, we find a novel class of active functional enhancers that are marked by H3K122ac but lack H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than was previously considered.

Compared with histone H3, acetylation of H4 tails has not been well studied, especially in mammalian cells. Yet, H4K16 acetylation is of particular interest because of its ability to decompact nucleosomes in vitro and its involvement in dosage compensation in flies. Here we show that, surprisingly, loss of H4K16 acetylation does not alter higher-order chromatin compaction in vivo in mouse embryonic stem cells (ESCs). As well as peaks of acetylated H4K16 and KAT8 histone acetyltransferase at the transcription start sites of expressed genes, we report that acetylation of H4K16 is a new marker of active enhancers in ESCs and that some enhancers are marked by H3K4me1, KAT8, and H4K16ac, but not by acetylated H3K27 or EP300, suggesting that they are novel EP300 independent regulatory elements. Our data suggest a broad role for different histone acetylation marks and for different histone acetyltransferases in long-range gene regulation.

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

Trithorax and polycomb group proteins are generally thought to antagonize one another. The trithorax family member MLL (myeloid/lymphoid or mixed-lineage leukemia) is presumed to activate Hox expression, counteracting polycomb-mediated repression. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, whose PWWP domain binds to H3K36me3, interacts with MLL and tethers MLL fusion proteins to HOXA9 in leukaemias. Here we show, unexpectedly, that Psip1/p75 regulates homeotic genes by recruiting not only MLL complexes, but also the polycomb group protein Bmi1. In Psip1−/− cells binding of Mll1/2, Bmi1 and the co-repressor Ctbp1 at Hox loci are all abrogated and Hoxa and Hoxd mRNA expression increased. Our data not only reveal a potential mechanism of action for Psip1 in the regulation of Hox genes but also suggest an unexpected interplay between proteins usually considered as transcriptional activators and repressors.

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2 nes ) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4 + CD8 + T-cell stage from which tumors initiate. Premalignant CD4 + CD8 + T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 singlemutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis.[Keywords: chromosome structure; condensin; genome instability; lymphoma; mitosis] Supplemental material is available for this article. Received May 23, 2016; revised version accepted September 15, 2016. Genome integrity is maintained by molecular machines that drive the duplication and segregation of the genome and by checkpoints that monitor for incorrect execution of these processes. As cells enter mitosis, chromosomes undergo profound structural changes, which are driven by topoisomerase II and condensins (Swedlow and Hirano 2003). This process removes catenations between sister chromatids, generates stiff rod-like structures that are competent for chromosome segregation, and is essential for genome propagation through the cell cycle in all eukaryotes.Condensins belong to the structural maintenance of chromosomes (SMC) complex family that also includes cohesin and SMC5/6. In eukaryotes, each SMC complex contains a heterodimer of SMC ATPase subunits, a single kleisin subunit, and additional accessory factors (Fig 1A;Losada and Hirano 2005;Nasmyth and Haering 2005). Within cohesin and condensin complexes, the C and N termini of kleisin interact with apposing SMC subunits to form a tripartite, asymmetric ring-like structure that can entrap DNA (Gruber et al. 2003;Onn et al. 2007;Nasmyth and Oliveira 2010;Cuylen et al. 2011; Piazza et al. Cold Spring Harbor Laboratory Press on May 10, 2018 -Published by genesdev.cshlp.org Downloaded from 2014). This mode of association has been proposed to allow SMC complexes to form topological linkages between chromosomes or different regions on the same DNA molecule.Metazoan genomes encode at least two distinct condensin complexes (Ono et al. 2003), whi...

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X  and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.