| সংক্ষিপ্ত: | <p>Enhancers are major determinants of tissue-specific gene expression. Although it is not entirely clear how enhancers activate target genes, nucleosomes at enhancers tend to have high levels of H3K27ac, which is deposited by the lysine acetyltransferases (KATs) CBP and P300. However, the extent to which histone acetylation is important for enhancer function is not fully elucidated. Recent work from the Milne laboratory (WIMM, University of Oxford) demonstrated that anchoring the transactivation domain of MYB (MYBTA) into a neutral chromatin environment can establish an enhancer-like element. Since MYB directly interacts with CBP/P300 we hypothesised this may be dependent upon the recruitment of CBP/P300 and subsequent deposition of histone acetylation. It was unclear whether this behaviour was unique to the genomic locus targeted and whether CBP/P300-mediated acetylation was necessary for this activity.</p>
<p>To investigate whether this effect was context dependent, I tethered MYBTA to a novel, neutral environment on a distinct chromosome using the TetR system in mouse embryonic stem cells. Surprisingly, despite activating the original locus, the enhancer activity of MYBTA did not extend to distal elements in this new context. This indicates that the enhancer forming ability of MYB is contingent upon the chromatin context within which it is bound. To test the necessity of CBP/P300 for MYB to generate an enhancer, I employed a proteolysis-targeting chimera (PROTAC), dCBP-1, to induce their degradation. This led to a reduction in H3K27ac genome-wide, coupled with transcriptional dysregulation. Despite a reduction in CBP/P300 and H3K27ac, MYBTA was still able to activate distal elements, but less potently. This suggests that CBP/P300 are not necessary for the full transcriptional activity of MYBTA at this locus.</p>
<p>Finally, to directly test the requirement of P300 KAT activity in enhancer function, I isolated the core domain of P300, containing the KAT domain and the bromodomain, and used this in the TetR chromatin targeting assay. This allowed me to examine the function of acetylation in the absence of the other CBP/P300 domains, while eliminating the complexity of transcription from nearby genes. The P300 core domain deposited H3K27ac and, interestingly, this was associated with activation of distal elements, analogous to an endogenous enhancer. Surprisingly, however, MYBTA and P300Core activated different elements within the same genomic region, despite being targeted to the same binding site. This suggests that MYB may drive transcription and enhancer function through additional or alternative mechanisms to P300-mediated acetylation.</p>
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