Abstract 4991: Dynamic reprogramming of the chromatin landscape in cancer: Studies in real time
Authors: Paakinaho Ville , Presman Diego M. , Swinstead Erin E. , Miranda Tina B. , Ball David A. , Karpova Tatiana S. , Hager Gordon L. 
Published: July 2017

Reprogramming of the chromatin landscape by sequence-specific transcription factors (TF) can influence in the prevalence and progression of several diseases, including cancers. Steroid receptors are a class of hormone-regulated TF that have a major impact on the progression of cancers. The link between the androgen receptor (AR) action in prostate cancer and the glucocorticoid (GR) and estrogen receptor (ER) action in breast cancer is well-known. Furthermore, other TFs such as the pioneer factor FoxA1 has been extensively linked to AR and ER action in these cancers. Although the action of these TF have been widely studied, the knowledge is almost exclusively based on population-based assays such as ChIP-seq and DHS-seq. Hence, the dynamic binding behavior of these factors in living cell remains unclear. For example, the classical pioneer factor model suggests long term static binding events between pioneer TFs, such as FoxA1, and chromatin. However, little is known on the dynamic action of FoxA1 in living cells.

We have examined the behavior of steroid receptors and cofactors, including FoxA1, at the single-molecule level in living cells. These factors were fused with HaloTag, and labeled with the bright and stable fluorophore JF549. Single-molecule tracking (SMT) of these proteins (using HILO microscopy) revealed a highly dynamic binding behavior of TFs. This supports transient rather than stable TF chromatin interactions. Specifically, two distinct binding populations have been observed for all factors tested; fast or slow stops. Fast stops represent genomic scanning of TF, while slow stops, with residence time of 6-14 sec, represent functional binding events at specific response elements. The vast majority of molecules at any given time are either diffusing or exhibit fast stops, while only a small percentage exhibit slow stops. However, hormone activation of AR, GR or ER results in an increase in the percentage of molecules with slow stops compared to the unstimulated state. Thus, our results indicate that on a single-molecule level, only a small proportion TFs are functionally bound at any given time.

In addition to affirming the general model that many TFs are highly dynamic in their chromatin binding activity, SMT experiments in live cells revealed a highly dynamic interaction of FoxA1 with chromatin in vivo. Unexpectedly, we also observed by ChIP-seq and DHS-seq analysis that at subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1. We propose a general model, dynamic assisted loading, wherein interactions between TFs and pioneer factors are highly dynamic. Our findings show that the action of key TFs in cancer development are more complex and dynamic in real time than previously reported.

Citation Format: Ville Paakinaho, Diego M. Presman, Erin E. Swinstead, Tina B. Miranda, David A. Ball, Tatiana S. Karpova, Gordon L. Hager. Dynamic reprogramming of the chromatin landscape in cancer: Studies in real time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2017-4991