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G M Fimia

Publications and source records attributed to G M Fimia.

17 recordsLinked to original sources
PubMed2001

Transcriptional cascades during spermatogenesis: pivotal role of CREM and ACT.

The gene CREM plays key physiological and developmental roles within the hypothalamic--pituitary--gonadal axis. We have previously shown that CREM is highly expressed in male postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. CREM regulates the expression of a number of post-meiotic genes involved in the process of spermiogenesis. Using homologous recombination we have generated CREM-mutant mice that display a complete block at the first step of spermiogenesis. The molecular mechanism by which CREM elicits its regulatory function involves ACT (Activator of CREM in Testis), a testis-specific coactivator constituted by a repeat of four and half LIM domains. ACT is coordinately expressed with CREM, associates with it and confers a powerful transcriptional activation function. It is able to bypass the classical requirement of CREM phosphorylation and recruiting of CBP.

Animals↗
PubMed2001

Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.

Metazoan genomes encode two related proteins, TBP and the TBP-like factor (TLF/TRF2), sharing a highly conserved saddle-like domain. TLF is highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The murine TLF gene has been inactivated using homologous recombination. TLF-/- mice are viable, but mutant male mice are sterile due to a late, complete arrest of spermiogenesis. In mutant animals, spermatogonia and spermatocytes develop normally, but round spermatids undergo apoptosis at step 7. Although the expression of the transcriptional activator CREM and many other postmeiotic genes was unaltered in TLF null mice, several spermiogenesis genes transcribed in late round spermatids appeared to be under TLF control. Hence, TLF is not required for embryonic development in the mouse but is essential for spermiogenesis.

Animals↗
PubMed2000

CREM, a master-switch of the transcriptional cascade in male germ cells.

In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. The CREB and CREM transcription factors are activated by phosphorylation of a key serine residue by kinase stimulated by cyclic AMP, calcium, growth factors and stress signals. Phosphorylation allows recruitment of CBP (CREB Binding Protein), a large co-activator that contacts the general transcriptional machinery. The CREM gene plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. CREM is highly expressed in postmeiotic cells upon a striking developmental switch regulated by the pituitary hormone FSH. CREM-mutant mice generated by homologous recombination reveal that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. Mutant male mice completely lack spermatozoa, a phenotype reminiscent of cases of human infertility. Interestingly, in male germ cells, CREM is not phosphorylated but associates with ACT, a member of the LIM-only class of proteins that has intrinsic transcriptional activity. Thus, in some circumstance, CREM can bypass the classical requirement for phosphorylation and association with CBP.

Animals↗
PubMed2000

A family of LIM-only transcriptional coactivators: tissue-specific expression and selective activation of CREB and CREM.

Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways. Regulation mediated by members of the CREB family has been linked to various physiological functions. Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP). However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized. We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis). ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP. Here we characterize a family of LIM-only proteins which share common structural organization with ACT. These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression. FHL proteins display different degrees of intrinsic activation potential. They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation. Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties. This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM. The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation.

Amino Acid Motifs↗
PubMed1999

CBP-independent activation of CREM and CREB by the LIM-only protein ACT.

Transcriptional activation by CREB and CREM requires phosphorylation of a serine residue within the activation domain (Ser 133 in CREB; Ser 117 in CREM) which as a result interacts with the coactivator CBP. The activator CREM is highly expressed in male germ cells and is required for post-meiotic gene expression. Using a two-hybrid screen, we have isolated a testis-derived complementary DNA encoding a protein that we term ACT (for activator of CREM in testis), a LIM-only protein which specifically associates with CREM. ACT is expressed coordinately with CREM in a tissue- and developmentally regulated manner. It strongly stimulates CREM transcriptional activity in yeast and mammalian cells and contains an intrinsic activation function. As ACT bypasses the classical requirements for activation, namely phosphorylation of Ser 117 and interaction with CBP, it represents a new route for transcriptional activation by CREM and CREB. ACT may define a previously undiscovered class of tissue-specific coactivators whose function could be specific for distinct cellular differentiation programmes.

Amino Acid Sequence↗
PubMed1999

Signaling routes to CREM and CREB: plasticity in transcriptional activation.

The CREB and CREM transcription factors are activated by phosphorylation of a key serine residue by kinases stimulated by cyclic AMP, Ca2+, growth factors and stress signals. Phosphorylation allows recruitment of CREB-binding protein (CBP), a large co-activator that contacts the general transcriptional machinery. Studies of the physiological roles played by CREB and CREM have uncovered novel routes of transcriptional activation. For example, in male germ cells CREM is not phosphorylated but associates with ACT, a member of the LIM-only class of proteins that has intrinsic transcriptional activity. Thus, in some circumstances, CREM can bypass the classical requirement for phosphorylation and association with CBP.

Animals↗
PubMed1999

Cyclic adenosine 3',5'-monophosphate(cAMP)/cAMP-responsive element modulator (CREM)-dependent regulation of cholesterogenic lanosterol 14alpha-demethylase (CYP51) in spermatids.

Lanosterol 14alpha-demethylase (CYP51) produces MAS sterols, intermediates in cholesterol biosynthesis that can reinitiate meiosis in mouse oocytes. As a cholesterogenic gene, CYP51 is regulated by a sterol/sterol-regulatory element binding protein (SREBP)-dependent pathway in liver and other somatic tissue. In testis, however, cAMP/cAMP-responsive element modulator CREMtau-dependent regulation of CYP51 predominates, leading to increased levels of shortened CYP51 mRNA transcripts. CREM-/- mice lack the abundant germ cell-specific CYP51 mRNAs in testis while expression of somatic CYP51 transcripts is unaffected. The mRNA levels of squalene synthase (an enzyme preceding CYP51 in cholesterol biosynthesis in testis of CREM-/- mice are unchanged as compared with wild-type animals, showing that regulation by CREMtau is not characteristic for all cholesterogenic genes expressed during spermatogenesis. The -334/+314 bp CYP51 region can mediate both the sterol/SREBP-dependent as well as the cAMP/CREMtau-dependent transcriptional activation. SREBP-1a from somatic cell nuclear extracts binds to a conserved CYP51-SRE1 element in the CYP51 proximal promoter. The cAMP-dependent transcriptional activator CREMtau from germ cell nuclear extracts binds to a conserved CYP51-CRE2 element while no SREBP-1 binding is observed in germ cells. The two regulatory pathways mediating expression of CYP51 describe this gene as a cholesterogenic gene (SREBP-dependent expression in liver and other somatic cells) and also as a haploid expressed gene (CREMtau-dependent expression in haploid male germ cells). While in somatic cells all genes involved in cholesterol biosynthesis are regulated coordinately by the sterol/SREBP-signaling pathway, male germ cells contain alternate routes to control expression of cholesterogenic genes.

Animals↗
PubMed1999

p53-independent apoptosis induced by muscle differentiation stimuli in polyomavirus large T-expressing myoblasts.

Abnormal proliferation signals, driven by cellular or viral oncogenes, can result in the induction of apoptosis under sub-optimal cell growth conditions. The tumor suppressor p53 plays a central role in mediating oncogene-induced apoptosis, therefore transformed cells lacking p53 are generally resistant to apoptosis-promoting treatments. In a previous work we have reported that the expression of polyomavirus large T antigen causes apoptosis in differentiating myoblasts and that this phenomenon is dependent on the onset of muscle differentiation in the absence of a correct cell cycle arrest. Here we report that polyomavirus large T increases the levels and activity of p53, but these alterations are not involved in the apoptotic mechanism. Apoptosis in polyomavirus large T-expressing myoblasts is not prevented by the expression of a p53 dominant-negative mutant nor it is increased by p53 over-expression. Moreover, forced differentiation induced through the over-expression of the muscle regulatory factor MyoD, leads to apoptosis without altering p53 function and, more significantly, even in a p53-null background. Our results indicate that apoptosis induced by the activation of muscle differentiation pathways in oncogene-expressing cells can occur in a p53-independent manner.

Animals↗
PubMed1998

Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R -/- mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R -/- mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Animals↗
PubMed1998

The activity of differentiation factors induces apoptosis in polyomavirus large T-expressing myoblasts.

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor beta or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G0(0) arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Animals↗
PubMed1996

Double-stranded internucleosomal cleavage of apoptotic DNA is dependent on the degree of differentiation in muscle cells.

Apoptotic cell death has been correlated to DNA fragmentation into discrete segments corresponding to the length of nucleosomal protected fragments of 180-200 base pairs or multiples of it. This DNA degradation has been ascribed to endonuclease activity that cleaves internucleosomally, thus giving rise to a ladder distribution upon electrophoretic migration. This strict correlation was, however, shown to have notable exceptions, since in some cases only single strand cleavage in the internucleosomal DNA regions has been observed (Tomei, D. L., Shapiro, P. J., and Cope, O. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 853-857). In the present work we show that mouse muscle cells, able to differentiate in vitro, if subjected to apoptosis present no DNA degradation into ladder form unless differentiation is previously induced. Furthermore, C3H/10T1/2 fibroblast cells, known to undergo apoptosis without DNA ladder formation, if converted to a myogenic program by MyoD expression, display internucleosomal DNA degradation upon induction of differentiation.

3T3 Cells↗
PubMed1995

A polyomavirus enhancer mutant confers ubiquitous high transcriptional efficiency to the SV40 late promoter.

To identify expression plasmids with high efficiency of transcription and with a broad tissue and cell range, we have constructed a recombinant vector combining the late SV40 promoter and the polyomavirus regulatory region derived from a mutant (PyNB11/1) which displays a very wide host range. We show that these recombinant enhancer-promoters are efficient drivers for heterologous gene transcription and expression in vitro in all mouse and human cells tested. The most active combination we identified contained the mutant enhancer (PyNB11/1) in the late orientation. This construct was able to promote a high efficiency of expression without significant fluctuation between cells of different tissutal origin or different differentiative stage. A possible interpretation of these results is discussed.

3T3 Cells↗
PubMed1994

Retinoblastoma antioncogene is involved in the inhibition of myogenesis by polyomavirus large T antigen.

The expression of polyomavirus large T antigen in stably transfected C2 myoblast cells inhibits terminal differentiation without inducing a transformed phenotype. In the present work, we report on the lifting of this inhibition by a mutation that prevents polyomavirus large T antigen from binding to the product of the retinoblastoma susceptibility gene (p105 RB). In contrast with cells containing wild-type large T, those with the Rb binding site mutant large T showed the same up-regulation of myosine heavy chain and myogenin mRNA expression as control cells. Furthermore, we correlate the cell cycle alteration induced by polyomavirus large T antigen expression with the inability of the cells to undergo terminal differentiation.

Animals↗
PubMed1992

Inhibition of in vitro myogenic differentiation by a polyomavirus early function.

In the present work we report on the role of a polyomavirus (Py) early function in interfering with both morphological and biochemical differentiation of the myogenic C2 cell line. The analysis of cell clones stably transfected with a plasmid carrying an ORI- Py genome showed that in the presence of the whole viral early region myogenesis is blocked and a transformed phenotype is evident. By using a plasmid that only encodes large-T function, the involvement of this individual early viral gene product was determined. Inhibition of myogenic differentiation by Py large T is proportional to the level of its expression. This inhibition does not appear to require alteration of cell growth properties. The analysis of muscle-specific functions expressed at different steps in the myogenic pathway showed that Py large T blocks the expression of terminal differentiation markers without altering the expression of the regulatory gene MyoD.

Animals↗
PubMed1992

Inhibition of in vitro muscle differentiation by the immortalizing oncogene py LT-ag.

The interference of Polyomavirus (Py) early functions with in vitro myogenic differentiation is the object of this study. Single cell analysis of C2 myogenic Py infected cells showed a mutual exclusion between Py early functions and muscle gene expression. The morphological and biochemical analysis of clones obtained from C2 cells stably transfected with a plasmid carrying an ORI- Py genome, showed that myogenesis is blocked and cells display the transformed phenotype. By using plasmids separately encoding Middle T or Large T functions, the involvement of individual early viral gene products was determined. Py Middle T alone does not inhibit myotube formation even though cells are morphologically transformed. Myogenic differentiation, on the other hand, is inhibited by Py Large T. This inhibition, which is proportional to the level of Py Large T expression, does not entail to require alteration of cell growth properties and acts by blocking the expression of myogenin and terminal differentiation markers without altering the expression of the regulatory gene MyoD.

Animals↗