The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification.

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.

Caspase-dependent regulation and subcellular redistribution of the transcriptional modulator YY1 during apoptosis.

The transcriptional regulator Yin Yang 1 (YY1) controls many aspects of cell behavior and is essential for development. We analyzed the fate of YY1 during apoptosis and studied the functional consequences. We observed that this factor is rapidly translocated into the cell nucleus in response to various apoptotic stimuli, including activation of Fas, stimulation by tumor necrosis factor, and staurosporine and etoposide treatment. Furthermore, YY1 is cleaved by caspases in vitro and in vivo at two distinct sites, IATD(12)G and DDSD(119)G, resulting in the deletion of the first 119 amino acids early in the apoptotic process. This activity generates an N-terminally truncated YY1 fragment (YY1Delta119) that has lost its transactivation domain but retains its DNA binding domain. Indeed, YY1Delta119 is no longer able to stimulate gene transcription but interacts with DNA. YY1Delta119 but not the wild-type protein or the caspase-resistant mutant YY1D12A/D119A enhances Fas-induced apoptosis, suggesting that YY1 is involved in a positive feedback loop during apoptosis. Our findings provide evidence for a new mode of regulation of YY1 and define a novel aspect of the involvement of YY1 in the apoptotic process.

PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation.

The proto-oncoprotein c-Myc functions as a transcriptional regulator that controls different aspects of cell behavior, including proliferation, differentiation, and apoptosis. In addition, Myc proteins have the potential to transform cells and are deregulated in the majority of human cancers. Several Myc-interacting factors have been described that mediate part of Myc's functions in the control of cell behavior. Here, we describe the isolation of a novel 150 kDa protein, designated PARP-10, that interacts with Myc. PARP-10 possesses domains with homology to RNA recognition motifs and to poly(ADP-ribose) polymerases (PARP). Molecular modeling and biochemical analysis define a PARP domain that is capable of ADP-ribosylating PARP-10 itself and core histones, but neither Myc nor Max. PARP-10 is localized to the nuclear and cytoplasmic compartments that is controlled at least in part by a Leu-rich nuclear export sequence (NES). Functionally, PARP-10 inhibits c-Myc- and E1A-mediated cotransformation of rat embryo fibroblasts, a function that is independent of PARP activity but that depends on a functional NES. Together, our findings define a novel PARP enzyme involved in the control of cell proliferation.

The human trithorax protein hASH2 functions as an oncoprotein.

Regulation of chromatin is an important aspect of controlling promoter activity and gene expression. Posttranslational modifications of core histones allow proteins associated with gene transcription to access chromatin. Closely associated with promoters of actively transcribed genes, trimethylation of histone H3 at lysine 4 (H3K4me3) is a core histone mark set by several protein complexes. Some of these protein complexes contain the trithorax protein ASH2 combined with the MLL oncoproteins. We identified human ASH2 in a complex with the oncoprotein MYC. This finding, together with the observation that hASH2 interacts with MLL, led us to test whether hASH2 itself is involved in transformation. We observed that hASH2 cooperates with Ha-RAS to transform primary rat embryo fibroblasts (REF). Furthermore, transformation of REFs by MYC and Ha-RAS required the presence of rAsh2. In an animal model, the hASH2/Ha-RAS-transformed REFs formed rapidly growing tumors characteristic of fibrosarcomas that, compared with tumors derived from MYC/Ha-RAS transformed cells, were poorly differentiated. This finding suggests that ASH2 functions as an oncoprotein. Although hASH2 expression at the mRNA level was generally not deregulated, hASH2 protein expression was increased in most human tumors and tumor cell lines. In addition, knockdown of hASH2 inhibited tumor cell proliferation. Taken together, these observations define hASH2 as a novel oncoprotein.

Overlap of the gene encoding the novel poly(ADP-ribose) polymerase Parp10 with the plectin 1 gene and common use of exon sequences.

We have recently identified PARP10 as a novel functional poly(ADP-ribose) polymerase. The gene encoding PARP10 is conserved in vertebrates but no orthologs were found in lower organisms. In addition to the poly(ADP-ribose) polymerase domain, PARP10 possesses several additional sequence motifs, including an RNA recognition motif and two ubiquitin interaction motifs. We characterized the murine genomic locus of the Parp10 gene. We noticed that 3' Parp10 sequences overlapped with the plectin 1 gene in a head-to-tail arrangement. Detailed analyses revealed that the two most 3' Parp10 exons (exons 10 and 11) are also used for plectin 1. While these two exons code for part of the poly(ADP-ribose) polymerase domain in Parp10, they are noncoding for plectin 1 due to the lack of appropriate start codons. Furthermore our findings suggest that at least one of the plectin 1 promoters is located within intron 9 of the Parp10 gene.

Stimulation of c-MYC transcriptional activity and acetylation by recruitment of the cofactor CBP.

The c-MYC oncoprotein regulates various aspects of cell behaviour by modulating gene expression. Here, we report the identification of the cAMP-response-element-binding protein (CBP) as a novel c-MYC binding partner. The two proteins interact both in vitro and in cells, and CBP binds to the carboxy-terminal region of c-MYC. Importantly, CBP, as well as p300, is associated with E-box-containing promoter regions of genes that are regulated by c-MYC. Furthermore, c-MYC and CBP/p300 function synergistically in the activation of reporter-gene constructs. Thus, CBP and p300 function as positive cofactors for c-MYC. In addition, c-MYC is acetylated in cells. This modification does not require MYC box II, suggesting that it is independent of TRRAP complexes. Instead, CBP acetylates c-MYC in vitro, and co-expression of CBP with c-MYC stimulates in vivo acetylation. Functionally, this results in a decrease in ubiquitination and stabilization of c-MYC proteins. Thus, CBP and p300 are novel functional binding partners of c-MYC.