Interactions of Chromatin Context,Binding Site Sequence Content,and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10⁻⁷) and correlated with stronger p53RE sequences (p<10⁻¹¹⁰) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.     

Androgen binding profiles of two distinct nuclear androgen receptors in Atlantic croaker (Micropogonias undulatus)

In the present study, the binding affinities of 28 androgens for two nuclear androgen receptors (AR), termed AR1 and AR2, in Atlantic croaker (Micropogonias undulatus) brain and ovarian tissues, respectively, were determined using competitive binding assays. The 5alpha-reduction of steroids, in general, increased the metabolite's binding affinity for AR2 while decreasing it for AR1. In addition, few androgens bound to AR1 with high affinity and modifications to the basic 3-ketone,4-ene,17beta-hydroxy structure of testosterone usually reduced its binding affinity for AR1. However, androgens with ketone groups at the 3- and 17-position bound with high affinity to AR1 provided that the androgen had either a 5alpha-reduced A-ring or a third ketone group at the 11-position. This suggests that there may be several high affinity conformations that AR1 can occupy depending upon whether an androgen possesses a ketone or a hydroxyl group at the 17-position. The binding of androgens to AR2 showed a more predictable pattern, 5alpha-reduced steroids bound better than 4-ene steroids and any changes to the basic 3-keto,17-hydroxy motif of 5alpha-dihydrotestosterone lowered the binding affinity of a steroid. However, these structural changes often caused only minor decreases in binding affinity, such that AR2 has a broader affinity for androgens and a greater affinity than AR1 for structurally diverse androgens. Widely different androgen binding affinities of AR1 and AR2 suggest that these two nuclear androgen receptors may mediate the physiological actions of different androgens in teleosts.