Investigating the molecular players involved in the control of transcriptional memory in metastasis

Metastasis is the principal cause of cancer-related mortality, yet the underlying molecular mechanisms supporting the invasion, dissemination, and dormancy of disseminated tumor cells (DTCs) remain unclear. In the metastasis cascade the DTCs encounter different microenvironmental stimuli and their fitness at distant sites is correlated to their phenotypic plasticity. By investigating the epigenetic state of a breast cancer xenograft-derived model we have highlighted that the activation of de novo oncogenic enhancers represents a hallmark of DTCs, fostering their cell plasticity. This metastatic chromatin context leads to the activation of a reinforced transcriptional response upon re-exposure to retinoic acid (atRA), thus establishing transcriptional memories. Of note, between the memory genes profiled by EU-RNA-seq, we found the master regulator SOX9, which plays a major role in supporting cancer dormancy. By employing an epigenome editing approach, we discovered that inhibiting the metastatic-enriched SOX9 enhancers was sufficient to repress the onset of RA-induced transcriptional memory. Moreover, the CDK8/CDK19 inhibition altered the robust transcriptional response of the memory genes. Despite this evidence, how these factors contribute specifically to the onset of transcriptional memory has not been elucidated yet (Michelatti et al., 2024). In the present work, we hypothesize that oncogenic SOX9 enhancers may act as docking sites for transcriptional condensates, favoring transcriptional memory. We first propose the coactivator p300 functions as a nucleating factor for the assembly of transcriptional condensates in response to atRA. In support, we showed that p300 is recruited at SOX9 enhancers upon treatment with atRA and, the transcriptional memory response is altered upon degradation of p300 during the chase period. To investigate the players involved and validate the putative role of p300, we employed an optogenetic approach to guide the assembly of different chromatin factors at SOX9 cis-regulatory elements (CREs). We first asked whether their action depended on their contribution to the assembly of transcriptional condensates, through the multiple interactions occurring among their intrinsically disordered regions (IDRs). We, therefore, guided the multi-clustering of the cofactors p300 and MLL4 IDRs using the SunTag-Cas9 strategy coupled with the optogenetic modules CRY2-CIB1 in a time-controlled manner. First, we verified the assembly of light-inducible chromatin condensates (LICC) and their ability to increase the steady state of SOX9 expression. We then found that the light-induced formation of LICC by IDRs of p300 phenocopies the molecular priming triggered by the treatment with RA. Of note, when we used the same approach by targeting MLL4 IDR, we did not observe the same transcriptional response. In conclusion, our work uncovers the contribution of the p300 IDRs to control SOX9 metastatic enhancers in the RA-mediated transcriptional memory, shedding light on the key molecular players that promote cancer dormancy.