CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

Abstract

NF‐Y binds a CCAAT motif found in many eukaryotic polymerase II‐dependent promoters. In the HLA‐DRA promoter it has been demonstrated that stereo‐specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild‐type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF‐Y binding. Interaction between NF‐Y and X box binding proteins was demonstrated by reciprocal co‐immunoprecipitation in the absence of DNA and co‐affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein‐DNA interactions and suggest that NF‐Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

“…There are also a number of additional possible phosphorylation sites throughout the protein, which is consistent with our previous finding that the CA-I activity can be phosphorylated (Sun et al, 1993). The CCAl clone has been mapped to a position near the bottom of chromosome 2 next to marker Athb-7 at 94.8 centimorgans on a yeast artificial chromosome-anchored physical map (Zachgo et al, 1996). We have also demonstrated that the CCAl protein is transported to nuclei in vivo, and this is consistent with its proposed function as a transcription factor.…”

“…The presence of a CCAAT box downstream of the conserved CCA1 binding sequence (Table 1) also raises the possibility that CCA1 could interact with a CCAAT-box binding protein. The CCAAT motif occurs in many eukaryotic promoters, and it has been proposed that factors binding to this sequence can facilitate recruitment of transcription factors binding upstream of this sequence (Wright et al, 1994). The proposed role in transcriptional activation of the protein(s) binding to the AAA A / C AATCTA region contrasts with our earlier proposal that CA-1 might act to repress transcription in dark-grown seedlings (Sun et al, 1993).…”

The S Box of Major Histocompatibility Complex Class II Promoters Is a Key Determinant for Recruitment of the Transcriptional Co-activator CIITA

“…It is also in agreement with in vivo footprint experiments, which have demonstrated that there is no clear footprint on the W/S box, whereas there is stable occupation of the X, X2, and Y boxes (61,62). Moreover, disruption of the S box in stably transfected reporter gene constructs does not alter the in vivo footprint pattern at the X, X2, and Y boxes (26). Finally, the precise spacing constraint also suggests that the S box serves a function distinct from the other cis-acting elements of MHCII promoters.…”

Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules

“…Finally, we propose that the NF-Y factor could organize the binding of the nearby factors. Such a situation is not unprecedented and previous report have pointed to the possibility that NF-Y might facilitate in vivo recruitment of upstream DNA binding transcription factors (Wright et al, 1994;Coustry et al, 1995). The resolution of this problem will await the puri®cation of the factor binding to the CCRE and the establisment of an in vitro assay to study these multiple interactions.…”