The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.

Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.

Pax5 controls the identity and development of B cells by repressing lineage-inappropriate genes and activating B-cell-specific genes. Here, we used genome-wide approaches to identify Pax5 target genes in pro-B and mature B cells. In these cell types, Pax5 bound to 40% of the cisregulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro-B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5-regulated genes in pro-B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro-B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro-B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B-cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. The EMBO Journal (2012) (Nutt and Kee, 2007). The helix-loop-helix protein E2A and the early B-cell factor EBF1 specify the B-cell lineage by activating the expression of B-lymphoid genes in pre-pro-B cells Treiber et al, 2010). Pax5 subsequently controls B-cell commitment at the transition to the pro-B cell stage by restricting the developmental potential of lymphoid progenitors to the B-cell lineage, as shown by the fact that Pax5-deficient pro-B cells are still able to differentiate into most hematopoietic cell types in vitro and in vivo (Nutt et al, 1999;Medvedovic et al, 2011). At the molecular level, Pax5 fulfills a dual role by repressing B-lineage-inappropriate genes to suppress alternative lineage options and by simultaneously activating B-cell-specific genes to promote B-cell development (Nutt et al, 1999;Medvedovic et al, 2011). Gene expression analyses of wild-type and Pax5-deficient pro-B cells identified 110 Pax5-repressed and 170 Pax5-activated genes, which code for key regulatory and structural proteins involved in transcriptional control, receptor signalling, adhesion, migration and immune function (Delogu et al, 2006;Schebesta et al, 2007;Pridans et al, 2008). Pax5 regulates these gene expression changes by inducing active chromatin at activated target genes and eliminating active chromatin at repressed genes in pro-B cells . Notably, Pax5 induces these chromatin and transcription changes by recruiting chromatin-remodelling, histone-modifying and basal transcription factor complexes to its target genes, which identifies Pax5 as an epigenetic regulator of B-cell commitment .Pax5 is expressed throughout B-cell development from pro-B cells in the bone marrow to mature B cells in peripheral lymphoid organs (Fuxa and Busslinger, 2007), where it plays an important role in the generation and function of distinct mature B-cell types (Horcher et al, 2001;Medvedovic et al, 2011). Pax5 is essential for maintaining the B-cell gene exp...

The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of different response pathways. The molecular mechanisms that discriminate between the distinct p53-responses have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by Actinomycin D and Etoposide treatment resulting in more growth arrested versus apoptotic cells respectively. We found that the genome-wide p53 DNA-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in global transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the genome-wide level of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46) and of Serine 15 (p53-pS15). Interestingly, the extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 remains highly similar. Moreover, our data suggest that following different chemotherapeutical treatments, the amount of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes. Our data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding and that post-translationally modified p53 can have distinct DNA-binding characteristics.