Monica Di Padova

Deacetylase Inhibitors Increase Muscle Cell Size by Promoting Myoblast Recruitment and Fusion through Induction of Follistatin

Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.

Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down‐regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che‐1, a novel human protein that interacts with hRPB11. Che‐1 possesses a domain of high homol‐ogy with Escherichia coli RNA polymerase σ‐factor 70 and SV40 large T antigen. In addition, we report that Che‐1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che‐1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the irans‐activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che‐1 may be part of transcription regulatory complex.—Fanciulli, M., Bruno, T, Di Padova, M., De Angelis, R., Iezzi, S., Iacobini, C, Floridi, A., Passananti, C. Identification of a novel partner of RNA polymerase II subunit 11, Che‐1, which interacts with and affects the growth suppression function of Rb. FASEB J. 14, 904–912 (2000)

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.

MyoD Acetylation Influences Temporal Patterns of Skeletal Muscle Gene Expression

MyoD is sufficient to initiate the skeletal muscle gene expression program. Transcription of certain MyoD target genes occurs in the early phases, whereas that of others is induced only at later stages, although MyoD is present throughout the differentiation process. MyoD acetylation regulates transcriptional competency, yet whether this post-translational modification is equally relevant for activation of all the MyoD targets is unknown. Moreover, the molecular mechanisms through which acetylation ensures that MyoD achieves its optimal activity remain unexplored. To address these two outstanding issues, we have coupled genome-wide expression profiling and chromatin immunoprecipitation in a model system in which MyoD or its nonacetylatable version was inducibly activated in mouse embryonic fibroblasts derived from MyoD-/-/Myf5-/- mice. Our results reveal that MyoD acetylation influences transcription of selected genes expressed at defined stages of the muscle program by regulating chromatin access of MyoD, histone acetylation, and RNA polymerase II recruitment.

Functional interaction of the subunit 3 of RNA polymerase II (RPB3) with transcription factor-4 (ATF4)

RPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial α subunit homodimer involved in promoter recognition. We previously employed the yeast two‐hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic α subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation.

The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin.

RNA polymerase II core subunit 3 (RPB3) is an α‐like core subunit of RNA polymerase II (pol II). It is selectively down‐regulated upon treatment with doxorubicin (dox). Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation. To this end, we have isolated human muscle RPB3interacting proteins by using yeast two‐hybrid screening. It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified. This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic α subunit. Although RPB3 contacts the basic helix‐loop‐helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4. Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction. We show that RPB3 expression is regulated during muscle differentiation. Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects. These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue‐specific transcription.

RNA polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

Here, we show that the subcellular localization of α-like RNA polymerase II core subunit 3 (RPB3) is regulated during muscle differentiation. We have recently demonstrated that the expression of RPB3 is regulated during muscle differentiation and that, inside RNA polymerase II (RNAP II), it is directly involved in contacting regulatory proteins such as the myogenic transcription factor Myogenin and activating transcription factor ATF4. We show for the first time, that RPB3, in addition to its presence and role inside the RNAP II core enzyme, accumulates in the cytoplasm of cycling myogenic cells and migrates to the nucleus upon induction of the differentiation program. Furthermore, using human RPB3 as bait in a yeast two-hybrid system, we have isolated a novel RPB3 cytoplasmic interacting protein, HCR. HCR, previously identified as α-helix coiled-coil rod homologue, is one of the psoriasis vulgaris (PV) candidate genes. In cycling myogenic C2C7 cells, we show that the RPB3 protein directly interacts with HCR within the cytoplasm. Finally, knocking down HCR expression by RNA interference, we demonstrate that HCR acts as cytoplasmic docking site for RPB3.