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  3. Handbook of Transcription Factor NF-kappaB - CRC Press Book
  4. Handbook of transcription factor NF-kappaB [2007]

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Original Research ARTICLE

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Written from a genome-based perspective, this volume provides an objective overview of transcription factor biochemistry. Topics include known transcription factor classes, origins and evolution of transcription factor types, and mechanisms of interaction with chromatin.

Transcription Factors

About This Item We aim to show you accurate product information. Manufacturers, suppliers and others provide what you see here, and we have not verified it. See our disclaimer. Specifications Series Title Subcellular Biochemistry. Customer Reviews. Write a review. See any care plans, options and policies that may be associated with this product. Email address. The positions of the Nkx2. D , in vitro -translated Nkx2. To understand how Nkx2. Thus, activation was stimulated 1. These results suggest that Nkx2. The rat insulin I and II genes share five of six nucleotide identity with the mouse insulin II genes in the core sequence of the Nkx2.

The data was compiled from at least six independently performed transfections. The Pdxspecific antibodies were found to selectively immunoprecipitate the control sequences of the IAPP , GK 36 , and pax-4 37 genes that bound Pdx-1 in vitro and were mutationally sensitive in transfection analyses Fig. Control reactions were performed as described in legend to Fig. These results confirm the in vitro studies that indicated that BETA2 was involved in pax-4 expression In addition, antisera to Pax6 Fig. Interestingly, the pax-4 promoter has been shown to contain a mutationally sensitive binding site that may be regulated by a factor in the Nkx2 family 39 , although the precise binding protein had not been identified.

Though assays such as in vitro binding, co-transfection with reporter constructs, and transgenic knock-out animals provide evidence for the involvement of a factor in transcriptional control, they do not prove this directly. This is especially problematic for factors, such as Pdx-1, that recognize relatively non-selective binding sites. As control region sequences are often several hundred bases in length, the presence of this motif is not uncommon and would not unequivocally demonstrate Pdx-1 regulation even if it were capable of binding to a cis -acting control element in vitro.

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Likewise, transfection experiments do not account for the native chromatin structure of endogenous genes, which may preclude physiological regulation by a factor that, nonetheless, can bind in vitro. The ChIP assay combines two highly specific techniques, antibody-specific protein-DNA precipitation and PCR amplification, to demonstrate a physical association between a transcription factor and specific control region sequences in intact cells.

The ChIP protocol can establish direct interactions of a transcription factor and a gene that are known to be functionally related and, importantly, can also be used to identify interactions with candidate target genes. Our demonstration of Pdx-1 occupancy of insulin control sequences corroborates a recent study that was published while our work was in progress Though there was evidence to suggest a direct role for Pdx-1 , 13 , 41 , BETA2 8 , 13 , and Pax6 9 in insulin gene expression, it has been unclear whether Nkx2.

Thus, the evidence supporting an involvement was based upon the loss of insulin expression in Nkx2. However, a control site for Nkx2. Based on the reported consensus Nkx2. Importantly, this site is mutationally sensitive in transfection assays, indicating that Nkx2. Collectively, our results demonstrate that Nkx2.

Handbook of Transcription Factor NF-kappaB - CRC Press Book

One would have expected that Nkx2. However, Nkx2. Interestingly, structure-function analysis of the Nkx2. As a consequence, it is possible that a co-factor expressed during pancreatic development or a stage-specific protein modification might interfere with the NK2-SD-mediated repressor function of Nkx2. In contrast, Nkx2. By extension of this reasoning, we presume that Nkx2. Although the Nkx. Having used the ChIP assay to demonstrate binding of Nkx2.

Previously, Pdx-1 had been shown to bind to its own promoter region in the ChIP assay 23 , Two distinct antisera raised against Pdx-1 were found to immunoprecipitate the IAPP and pax-4 control sequences. However, whereas we observed a clear interaction of Pdx-1 and the GK gene using antisera to both the N and C termini of Pdx-1, another group was unable to detect this association using a different Pdx-1 antiserum This difference highlights an important limitation of the ChIP assay, which despite the power of this technique to firmly establish a physical association between a transcription factor and a control region with immunoprecipitating antisera, a negative result does not provide definitive evidence for the lack of such interactions.

There are two likely explanations for these negative results.

Handbook of transcription factor NF-kappaB [2007]

The microenvironment of the GLUT2 control region might preclude the exposure of Pdx-1 antigenic determinants required for immunoprecipitation, possibly due to interactions with adjacent binding proteins. Alternatively, it is possible that Pdx-1 does not directly regulate GLUT2 transcription or does so through elements that are located outside of the analyzed region.

Both studies focused on the proximal region of the GLUT2 promoter found to bind Pdx-1 in vitro data not shown and Ref. How each of these factors precisely mediates control within the context of the insulin, pax-4 , IAPP, and GK transcription units is still unclear. We thank Drs.


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Chris Wright and Joel Habener for generously providing the N-terminal amino acids 1—75 and C-terminal amino acids — Pdx-1 antiserum, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. You'll be in good company. Journal of Lipid Research.

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see url Figure 2 Nkx2. Figure 3 Nkx2.