Stable Expression of a Dominant Negative Mutant of CCAAT Binding Factor/NF-Y in Mouse Fibroblast Cells Resulting in Retardation of Cell Growth and Inhibition of Transcription of Various Cellular Genes

Hu, Qianghua; Maity, Sankar N.

doi:10.1074/jbc.275.6.4435

Cited by 63 publications

(78 citation statements)

References 46 publications

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“…In agreement with this, the levels of NF-Y activity in the cells strongly influences cell proliferation. It has been reported that inhibition of NF-YA expression blocks cell cycle progression in G1 and G2 (Hu and Maity, 2000) and the knock out of the NF-YA subunit in mice leads to embryo lethality (Bhattacharya et al, 2003). Thus, there may be specific mechanisms for limiting free NF-Y levels, failure of which would compromise cell survival and/or homeostasis.…”

Section: Discussionmentioning

confidence: 99%

“…Knock-out mice clearly demonstrates that NF-Y dependent transcription is essential during early mouse development (Bhattacharya et al, 2003). The NF-Y function in proliferation is also demonstrated by the inhibition of DNA binding by endogenous NF-Y, resulting in retardation of fibroblast growth, which is brought about by overexpression of a dominant negative mutant of the NF-YA subunit (Hu and Maity, 2000). The differential expression of NF-YA, altering NF-Y CCAAT-binding activity, has been observed in several cell lines and tissues both during cell cycle progression and under specific conditions.…”

Section: Introductionmentioning

confidence: 99%

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Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

Manni¹,

Caretti

Artuso³

et al. 2008

MBoC

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Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity. INTRODUCTIONThe CCAAT-binding transcription factor NF-Y is a heteromeric protein composed of three subunits, NF-YA, -YB, and -YC, all necessary for CCAAT binding (Mantovani, 1999). The NF-Y complex supports the basal transcription of a class of regulatory genes responsible for cell cycle progression, among which are mitotic cyclin complexes (Zwicker et al., 1995a,b;Bolognese et al., 1999;Farina et al., 1999;Korner et al., 2001;Gurtner et al., 2003Gurtner et al., , 2008Di Agostino et al., 2006). Knock-out mice clearly demonstrates that NF-Y dependent transcription is essential during early mouse development (Bhattacharya et al., 2003). The NF-Y function in proliferation is also demonstrated by the inhibition of DNA binding by endogenous NF-Y, resulting in retardation of fibroblast growth, which is brought about by overexpression of a dominant negative mutant of the NF-YA subunit (Hu and Maity, 2000). The differential expression of NF-YA, altering NF-Y CCAAT-binding activity, has been observed in several cell lines and tissues both during cell cycle progression and under specific conditions. NF-YA expression is modulated during the cell cycle, being high in G1, further increasing in S, and then decreasing in the G2/M phase (Bolognese et al., 1999). Reduction of NF-YA expression has been reported in IMR-90 fibroblasts after serum deprivation (Chang et al., 1994) and in human monocytes (Marziali et al., 1997). The NF-YA protein is not expressed in terminally differentiated C2C12 muscle cells and adult skeletal muscle tissues (Farina et al., 1999;Gurtner et al., 2003Gurtner et al., , 2008. These observations show that levels of NF-YA dictate the function of the NF-Y complex. Interestingly, control of NF-YA accumulation is posttranscriptional, because NF-YA mRNA is relatively constant in growing and differentiated cells (Bolognese et al., 1999;Farina et al., 1999). Yet, the mechanisms regulating the stability of the NF-YA protein remain unknown.The covalent addition of ubiquitin, a 76-amino acid protein highly conserved among eukaryotes, is an increasingly common posttranslation modification that controls both the expression and the activity of numerous proteins in the eukaryotic cell (Hershko and Ciechanover, 1998;Ciechanover et al., 2000). The ubiquitin-proteasome pathway is composed of the ubiquitin-conjugating system and the 26S proteasome; the latter contain...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

Manni¹,

Caretti

Artuso³

et al. 2008

MBoC

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“…Recently, it was reported that a dominant-negative mutant form of YA, which contains amino-acid substitutions within the DNA-binding domain and inhibits DNA-binding activity of NF-Y in vitro, produces a delayed induction of the S phase in quiescent fibroblast cells after serum stimulation (Hu and Maity, 2000). Therefore, this YA mutant and YA-aa exhibited identical behavior in vitro DNA-binding assays, inhibiting DNA-binding activity of NF-Y in a dominant-negative manner.…”

Section: Discussionmentioning

confidence: 99%

Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions

et al. 2004

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We previously reported that cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y transcription factor and this phosphorylation is essential for DNA binding of NF-Y. In this study, we examined the effects of a phosphorylationdeficient mutant form of YA, YA-aa, in which the two serine residues are replaced with alanine, on the cell cycle and expression of the NF-Y target genes. Transient transfection assays show that YA-aa inhibits transcription from the NF-Y target promoters, such as cdc2, cyclin A, and cdc25C. Moreover, this inhibitory function of YA-aa can be suppressed by the expression of wild-type YA, implying that YA-aa inhibits transcription of those NF-Y target genes by inactivating wild-type YA. Since NF-Y target genes include the cell cycle-regulatory genes that ensure orderly progression of the cell cycle, we examined the effects of YA-aa in cell cycle progression. We constructed a recombinant adenovirus encoding YA-aa and found that YA-aa expression leads to repression of cell cycle-regulatory genes, such as cyclin A, RNR R2, DNA polymerase a, cdc2, cyclin B, and cdc25C. Consistently, YA-aa expression results in the inactivation of both cdc2 and cdk2. Furthermore, cell cycle analysis reveals that YA-aa induces cell cycle arrest at both G1 and G2/M. These results suggest that cdk2-dependent phosphorylation of NF-Y is essential for the expression of the cell cycle-regulatory genes and therefore for cell cycle progression at both G1/S and G2/M.

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“…49,78,79 Importantly, removal of the NF-YA (CBF-B) Q-rich domain generates a dominant-negative mutant, which affects the activity of CCAAT promoters upon transfection. 80 Co-transfections of the NF-Y trimer with TFs binding the loci neighboring CCAAT boxes in mammalian and Drosophila cells synergistically activate transcription of CCAAT promoters. 52,81,82 Finally, transfections of recombinant proteins in which NF-YA was linked to a cell-penetrating TAT peptide activate endogenous CCAAT genes but not CCAAT-less units.…”

Section: Nf-y Is the Sequence-specific Ccaat Factormentioning

confidence: 99%

Targeting the Y/CCAAT box in cancer: YB-1 (YBX1) or NF-Y?

Dolfini

Mantovani

2013

Cell Death Differ

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The Y box is an important sequence motif found in promoters and enhancers containing a CCAAT box -one of the few elements enriched in promoters of large sets of genes overexpressed in cancer. The search for the transcription factor(s) acting on it led to the biochemical purification of the nuclear factor Y (NF-Y) heterotrimer, and to the cloning -through the screening of expression libraries -of Y box-binding protein 1 (YB-1), an oncogene, overexpressed in aggressive tumors and associated with drug resistance. These two factors have been associated with Y/CCAAT-dependent activation of numerous growth-related genes, notably multidrug resistance protein 1. We review two decades of data indicating that NF-Y ultimately acts on Y/CCAAT in cancer cells, a notion recently confirmed by genome-wide data. Other features of YB-1, such as post-transcriptional control of mRNA biology, render it important in cancer biology.

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Stable Expression of a Dominant Negative Mutant of CCAAT Binding Factor/NF-Y in Mouse Fibroblast Cells Resulting in Retardation of Cell Growth and Inhibition of Transcription of Various Cellular Genes

Cited by 63 publications

References 46 publications

Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions

Targeting the Y/CCAAT box in cancer: YB-1 (YBX1) or NF-Y?

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