Posttranslational modifications play a key role in recruiting chromatin remodeling and modifying enzymes to specific regions of chromosomes to modulate chromatin structure. Alc1 (amplified in liver cancer 1), a member of the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, is encoded by an oncogene implicated in the pathogenesis of hepatocellular carcinoma. Here we show that Alc1 interacts transiently with chromatin-associated proteins, including histones and the poly(ADP-ribose) polymerase Parp1. Alc1 ATPase and chromatin remodeling activities are strongly activated by Parp1 and its substrate NAD and require an intact macrodomain capable of binding poly(ADP-ribose). Alc1 is rapidly recruited to nucleosomes in vitro and to chromatin in cells when Parp1 catalyzes PAR synthesis. We propose that poly(ADPribosyl)ation of chromatin-associated Parp1 serves as a mechanism for targeting a SNF2 family remodeler to chromatin.Alc1 ͉ chromatin remodeling enzyme ͉ macrodomain ͉ poly-(ADP-ribose) polymerase ͉ Snf2-like ATPase I n eukaryotic cells, chromosomal DNA is packaged into nucleosomes, which are in turn folded into higher order nucleosome arrays in chromatin fibers. This packaging allows the Ϸ2 m of DNA that make up the human genome to fit into nuclei with diameters on the order of 2-6 m; however, it also blocks access to DNA of the machinery responsible for transcription, replication, and DNA repair. Eukaryotic organisms have evolved a set of chromatin modifying and remodeling enzymes that alter the structure of chromatin to control accessibility to the machineries responsible for DNA replication and repair and for transcription. These enzymes have been shown to be targeted to regions of modified chromatin by such domains as bromodomains, which can bind acetylated histones, or chromodomains, tudor domains, or MBT domains, which can interact with methylated histones (1-4).ALC1 (amplified in liver cancer 1), alternatively known as CHD1L, is a member of the SNF2 superfamily of ATPases, some of which function as chromatin remodeling enzymes (5-7). Sequence alignments suggest that Alc1 is similar to chromatin remodeling ATPases Snf2, Iswi, and Chd1, which have been implicated in transcription, DNA repair, and replication (7). Alc1 lacks identifiable chromo-, bromo-, tudor-, MBT, or other domains known to have chromatin targeting functions. Instead, it contains a carboxy-terminal macrodomain. Macrodomains have been shown through biochemical and structural analyses to bind ADP-ribose (8).Over 50% of human hepatocellular carcinoma (HCC) patients contain a chromosomal amplification at 1q21, which includes the ALC1 gene (9-11). Alc1-overexpressing cells exhibit increased colony formation in soft agar and increased tumorigenicity in nude mice (11), suggesting that ALC1 functions as an oncogene.While mounting evidence points to a potential role for Alc1 in oncogenesis, the molecular function of the Alc1 ATPase has not been studied. Here, we show that Alc1 is a chromatin remodeling enzyme that is recruited to nucleosom...

Here, we report generation and characterization of Disrupted-InSchizophrenia-1 (DISC1) genetically engineered mice as a potential model for major mental illnesses, such as schizophrenia. DISC1 is a promising genetic risk factor for major mental illnesses. In this transgenic model, a dominant-negative form of DISC1 (DN-DISC1) is expressed under the ␣CaMKII promoter. In vivo MRI of the DN-DISC1 mice detected enlarged lateral ventricles particularly on the left side, suggesting a link to the asymmetrical change in anatomy found in brains of patients with schizophrenia. Furthermore, selective reduction in the immunoreactivity of parvalbumin in the cortex, a marker for an interneuron deficit that may underlie cortical asynchrony, is observed in the DN-DISC1 mice. These results suggest that these transgenic mice may be used as a model for schizophrenia. DN-DISC1 mice also display several behavioral abnormalities, including hyperactivity, disturbance in sensorimotor gating and olfactory-associated behavior, and an anhedonia/ depression-like deficit.

Summary Promoter proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of SuperElongationComplexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is currently a major unanswered question. Here, we present evidence for a role of human Mediator subunit Med26 in this process. We identify in the conserved N-terminal domain of Med26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that Med26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

The proteasome degrades proteins modified by polyubiquitylation, so correctly controlled ubiquitylation is crucial to avoid unscheduled proteolysis of essential proteins. The mechanism regulating proteolysis of RNAPII has been controversial since two distinct ubiquitin ligases (E3s), Rsp5 (and its human homologue NEDD4) and Elongin-Cullin complex, have both been shown to be required for its DNA-damage-induced polyubiquitylation. Here we show that these E3s work sequentially in a two-step mechanism. First, Rsp5 adds mono-ubiquitin, or sometimes a ubiquitin chain linked via ubiquitin lysine 63 that does not trigger proteolysis. When produced, the K63 chain can be trimmed to mono-ubiquitylation by an Rsp5-associated ubiquitin protease, Ubp2. Based on this mono-ubiquitin moiety on RNAPII, an Elc1/Cul3 complex then produces a ubiquitin chain linked via lysine 48, which can trigger proteolysis. Likewise, for correct polyubiquitylation of human RNAPII, NEDD4 cooperates with the ElonginA/B/C-Cullin 5 complex. These data indicate that RNAPII polyubiquitylation requires cooperation between distinct, sequentially acting ubiquitin ligases, and raise the intriguing possibility that other members of the large and functionally diverse family of NEDD4-like ubiquitin ligases also require the assistance of a second E3 when targeting proteins for degradation.elongin ͉ NEDD4 ͉ Rsp5 ͉ ubiquitylation P rotein ubiquitylation plays a crucial role in virtually all cell regulatory pathways. Mono-ubiquitylation commonly alters the activity of the target protein, or tags it for interaction with other factors, while the effect of polyubiquitylation depends on the type of ubiquitin chain being added. Ubiquitin lysine 48 (K48) chains most often result in degradation of the target protein by the proteasome, whereas other chains, such as those occurring through K63, are typically signals for proteolysisindependent pathways (1, 2).One interesting substrate for protein ubiquitylation is RNA-PII, which transcribes all protein-encoding genes in eukaryotes. Ubiquitylation and degradation of RNAPII was first thought to occur specifically in response to DNA damage (3-5), but more recent experiments have shown that RNAPII arrested during transcript elongation as a result of other transcription obstacles is also prone to ubiquitylation and degradation (6). Thus, degradation of RNAPII may be a ''last resort,'' used to clear active genes of persistently arrested RNAPII elongation complexes (6-9). Interestingly, the proteasome is nuclear and can be found on the coding region of genes by chromatin-immunoprecipitation (10), so RNAPII proteolysis may well occur on the DNA.We have reconstituted RNAPII ubiquitylation in vitro with highly purified, physiologically relevant yeast, or human, ubiquitylation factors, respectively (6,11,12). The yeast HECT E3 Rsp5 binds RNAPII via the flexible C-terminal repeat domain (CTD) of the Rpb1 subunit (13), but modifies the polymerase in the main body of the Rpb1 subunit (6,14). Mutation of RSP5 (rsp5-1; temperature-sensitiv...

Oxidative lesions represent the most abundant DNA lesions within the cell. In the present study, we investigated the impact of the oxidative lesions 8-oxoguanine, thymine glycol and 5-hydroxyuracil on RNA polymerase II (RNA pol II) transcription using a well-defined in vitro transcription system. We found that in a purified, reconstituted transcription system, these lesions block elongation by RNA pol II to different extents, depending on the type of lesion. Suggesting the presence of a bypass activity, the block to elongation is alleviated when transcription is carried out in HeLa cell nuclear extracts. By purifying this activity, we discovered that TFIIF could promote elongation through a thymine glycol lesion. The elongation factors Elongin and CSB, but not TFIIS, can also stimulate bypass of thymine glycol lesions, whereas Elongin, CSB and TFIIS can all enhance bypass of an 8-oxoguanine lesion. By increasing the efficiency with which RNA pol II reads through oxidative lesions, elongation factors can contribute to transcriptional mutagenesis, an activity that could have implications for the generation or progression of human diseases.