Reduced miR-204 expression facilitates the excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells characteristic of human pulmonary arterial hypertension.

SUMMARY The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here, we identified MBTD1 as a new stable subunit of the complex and gleaned intriguing insights into TIP60’s function. Harboring a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double strand breaks by homologous recombination. Interestingly, the non-homologous end joining factor 53BP1 engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub, and it was postulated that Tip60-dependent acetylation of H4 regulates this binding. Our findings now indicate that the TIP60 complex is a potent regulator of DNA damage repair pathways in part by targeting the same histone mark as 53BP1. In addition, deposition of H2AK15ub by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex which regulates 53BP1-dependent repair pathway selection through incompatible bivalent binding and modification of chromatin.

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Because microRNAs have been recently implicated in the regulation of cell proliferation and apoptosis, we hypothesized that these regulatory molecules might be implicated in the etiology of PAH. In this study, we show that miR-204 expression in PASMCs is down-regulated in both human and rodent PAH. miR-204 down-regulation correlates with PAH severity and accounts for the proliferative and antiapoptotic phenotypes of PAH-PASMCs. STAT3 activation suppresses miR-204 expression, and miR-204 directly targets SHP2 expression, thereby SHP2 up-regulation, by miR-204 down-regulation, activates the Src kinase and nuclear factor of activated T cells (NFAT). STAT3 also directly induces NFATc2 expression. NFAT and SHP2 were needed to sustain PAH-PASMC proliferation and resistance to apoptosis. Finally, delivery of synthetic miR-204 to the lungs of animals with PAH significantly reduced disease severity. This study uncovers a new regulatory pathway involving miR-204 that is critical to the etiology of PAH and indicates that reestablishing miR-204 expression should be explored as a potential new therapy for this disease. role of miR-204 in the etiology of PAH. Interestingly, using in silico and microarray gene expression analyses, we observed that among the 461 predicted targets of miR-204 (TargetScan 5.1), only 165 were increased by artificial miR-204 inhibition in control human PASMCs (n = 2 patients; Fig. S1 C). In accordance with the pro-proliferative and antiapoptotic phenotypes seen in PAH, several Src-STAT3-and NFAT-related genes were identified (Fig. S1 C). miR-204 expression is decreased in human PAH and correlates with PAH severityTo investigate the expression pattern of miR-204 in normal and pulmonary hypertensive lungs, we examined miR-204 expression levels in (a) lung biopsies from 8 individuals with nonfamilial PAH compared with biopsies from 8 individuals without pulmonary hypertension, (b) lungs from 6 mice with hypoxia-induced pulmonary hypertension compared with 5 control littermates, and (c) lungs from 5 rats with monocrotaline (MCT)-induced pulmonary hypertension compared with 10 control littermates ( Fig. 1 A). We found decreased levels of miR-204 in human and rodent pulmonary hypertensive lung tissues compared with normotensive lung samples. To characterize whether down-regulated miR-204 levels were specific to the lung in rats with pulmonary hypertension, we compared organ-specific levels of miR-204 between normal and pulmonary hypertensive rats (Fig. 1 B). Even if we were able to detect minimal amounts of miR-204 in most organs, miR-204 levels were only down-regulated in the lung and PAs but not in the aorta, liver, heart, and kidney in rats 3 wk after MCT injection (pulmonary hypertensive rats) compared with non-pulmonary hypertensive rats (Fig. 1 B).To test whether miR-204 down-regulation correlated with disease progression, we studied humans, mice, a...

SUMMARY The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity towards its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification cross-talk within a single protein complex.

The instability of a breast cancer subtyping scheme like PAM50 could have important consequences in clinical management of patients. AIMS is a fully stable and robust subtyping scheme that recapitulates PAM50.

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.Alternative splicing is a critical process that ensures the production of a multitude of proteins from a limited set of mammalian genes (7, 41). Alternative splicing decisions are regulated by a large collection of RNA-binding proteins (RBPs) that bind to pre-mRNAs in the nucleus (5). The heterogeneous nuclear ribonucleoparticle (hnRNP) proteins are among the most abundant of such proteins, and more than 20 of them have been characterized and given alphabetical names based on size from hnRNP A1 to hnRNP U (17). These proteins have been implicated in a variety of biological processes including telomere biogenesis, translation, and RNA stability, and several (e.g., hnRNP A1, A2, F, H, I [PTB], G, and L) have documented roles in splicing (34, 38). hnRNP A1 has been implicated in the splicing control of many genes, including the A1 gene itself, the caspase-2 gene, c-src, and the SMN2 gene (12), and several exons of human immunodeficiency virus type 1; the very similar hnRNP A2 protein (68% identity) appears to display comparable activity (4,10,29,42). While the related hnRNP F and H proteins play a role in splicing control of many genes, including c-src, Bcl-x, cystathionine ␤-synthase, and several HIV alternative exons (38), it is unclear whether F and H have completely redundant activities. hnRNP I (PTB) is another well-known splicing regulator that has been mostly associated with splicing repression (54,59). A recent global analysis of PTB and its neural paralogue nPTB has revealed their role in the control of murine neuron-specific splicing (8). hnRNP G (RBM-X) has been implicated in the splicing...

Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.