The Mechanism Underlying The Coordinated Regulation Of BCL2 And NOXA Genes By The Long-range Regulatory Element Mbr

The gene expression of eukaryotic cells is regulated by diverse regulatory elements that widely spread over the genome. Each single regulatory element may regulate multiple genes and a single gene may also be regulated by multiple regulatory elements. Thus, a sophisticated 3-dimensional transcription-regulating network is formed at the chromatin level, which ensures accurate transcriptional regulation of genes. The function of distal regulatory elements is closely related to those high-order structures of chromatin. The change of high-order structures affects the interaction between distal elements and their target genes, and further the gene transcription level. However, in different cellular activities, molecular mechanism underlying the coordinated regulation of different target genes by long-range regulatory element through the dynamic architecture is unclear.The fate of cells is determined by the reaction of cells to apoptotic signals. BCL2 gene family members play a crucial role in the regulation of apoptosis signaling pathways. The accurate regulation at the transcriptional level and coordination of different members of the apoptotic signal response are critical for maintaining the steady state of cells and tissues. The NOXA gene and BCL2 gene are both members of BCL2 family, yet the former is pro-apoptotic while the later is anti-apoptotic. In response to apoptotic stimuli, the NOXA transcription level increased meanwhile the BCL2 level decreased. Our previous study has revealed that the major breakpoint region(mbr) located within 3?-UTR of the BCL2 gene is a positive regulatory element. It significantly enhanced transcriptional activity of the BCL2 promoter over 200 kb long distance. This function was dependent on the SATB1-mediated interaction between the mbr and BCL2 gene promoter. We also found that NOXA gene located 3.4Mb downstream of the BCL2 gene was another target gene of the mbr element. The interaction between mbr and NOXA promoter was positively correlated with NOXA transcription but negatively correlated with BCL2 transcription. In early response of cells to apoptotic stimuli mbr-NOXA interaction was significantly increased and NOXA transcription was dramatically enhanced, while mbr-BCL2 interaction was reduced which resulted in decrease in BCL2 transcription. As our research depicts, mbr could selectively enhance and further coordinate the expression of BCL2 and NOXA genes due to a dynamic change from mbr-BCL2 loop to mbr-NOXA loop. However,the underlying molecular mechanism leading to such transition of the chromatin loops and its relationship with the mbr regulatory function is still to be elucidated.Special AT-rich sequence binding protein 1(SATB1) belongs to a class of transcription factors that function as a landing platform. SATB1 participates in the dynamic change of chromatin loops and the long range regulation. SATB1 and its homeotic protein compete to bind with certain chromatin to regulate transcription of target genes. CDP is a homeodomain protein that appears to compete with transcriptional activating proteins for binding to the promoter regions of various genes. Conbinding the function of SATB1 and what we have known, we hypothesized that change in SATB1 level was the key factor that launches dynamic transition between mbr-BCL2 and mbr-NOXA interactions during cell apoptosis response. Apoptotic stimulation-induced degradation of SATB1 resulted in disassociation of SATB1 from mbr and alteration of protein complex binding on mbr, which impaired mbr-BCL2 interaction and enhances mbr-NOXA interaction. As a result, NOXA expression was enhanced while BCL2 expression is suppressed.This study employs a series of experiments which focus on analysing the mechanisms connecting several vital processes: the degradation of SATB1, the change of mbr binding protein, the conversion of high-order chromatin structure from mbr-BCL2 to mbr-NOXA, and the coordinated long-distance regulation of BCL2 and NOXA gene to verify our hypothesis. Methods such as chromatin conformation capture(3C), chromatin immunoprecipitation(ChIP) assays and real-time PCR are used throughout the study. Otherwise, using method of 4C, we found multiple potential target genes besides BCL2 family menbers and numerous potential regulatory elements.Our data provides new clues for understanding the molecular mechanism undering the coordinated regulation of different genes by the same long-range regulatory element for the first time, and also provide a new insight to explain the role of high-order chromatin structures in coordinated expression of functional correlated genes in eukaryotic cells.