Myc-binding-site recognition in the human genome is determined by chromatin context.

Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo.

Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive.

Eukaryotic genomes are organized into active (euchromatic) and inactive (heterochromatic) chromatin domains. Post-translational modifications of histones (or 'marks') are key in defining these functional states, particularly in promoter regions. Mutual regulatory interactions between these marks--and the enzymes that catalyse them--contribute to the shaping of this epigenetic landscape, in a manner that remains to be fully elucidated. We previously observed that asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) counter-correlates with di- and tri- methylation of H3 lysine 4 (H3K4me2, H3K4me3) on human promoters. Here we show that the arginine methyltransferase PRMT6 catalyses H3R2 di-methylation in vitro and controls global levels of H3R2me2a in vivo. H3R2 methylation by PRMT6 was prevented by the presence of H3K4me3 on the H3 tail. Conversely, the H3R2me2a mark prevented methylation of H3K4 as well as binding to the H3 tail by an ASH2/WDR5/MLL-family methyltransferase complex. Chromatin immunoprecipitation showed that H3R2me2a was distributed within the body and at the 3' end of human genes, regardless of their transcriptional state, whereas it was selectively and locally depleted from active promoters, coincident with the presence of H3K4me3. Hence, the mutual antagonism between H3R2 and H3K4 methylation, together with the association of MLL-family complexes with the basal transcription machinery, may contribute to the localized patterns of H3K4 tri-methylation characteristic of transcriptionally poised or active promoters in mammalian genomes.

Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response.

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using E(mu)-myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/- mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF-p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from-but not contradictory with-its role within the ARF-p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

Analysis of Myc-induced histone modifications on target chromatin.

The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression) of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks) at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP) to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1), incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.