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SummaryDefining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.

Summary Structural and functional details of the N-terminal activation function 1 (AF1) of most nuclear receptors are poorly understood due to the highly dynamic intrinsically disordered nature of this domain. A hydrogen/deuterium exchange (HDX) mass-spectrometry-based investigation of TATA box-binding protein (TBP) interaction with various domains of progesterone receptor (PR) demonstrate that agonist-bound PR interaction with TBP via AF1 impacts the mobility of the C-terminal AF2. Results from HDX and other biophysical studies involving agonist- and antagonist-bound full-length PR and isolated PR domains reveal the molecular mechanism underlying synergistic transcriptional activation mediated by AF1 and AF2, dominance of PR-B isoform over PR-A, and the necessity of AF2 for full AF1-mediated transcriptional activity. These results provide a comprehensive picture elaborating the underlying mechanism of PR-TBP interactions as a model for studying nuclear receptor (NR)-transcription factor functional interactions.

The N-terminal domain (NTD) of steroid receptors harbors a transcriptional activation function (AF1) that is composed of an intrinsically disordered polypeptide. We examined the interaction of the TATA-binding protein (TBP) with the NTD of the progesterone receptor (PR) and its ability to regulate AF1 activity through coupled folding and binding. As assessed by solution phase biophysical methods, the isolated NTD of PR contains a large content of random coil, and it is capable of adopting secondary α-helical structure and more stable tertiary folding either in the presence of the natural osmolyte trimethylamine-N-oxide or through a direct interaction with TBP. Hydrogen-deuterium exchange coupled with mass spectrometry confirmed the highly dynamic intrinsically disordered property of the NTD within the context of full-length PR. Deletion mapping and point mutagenesis defined a region of the NTD (amino acids 350–428) required for structural folding in response to TBP interaction. Overexpression of TBP in cells enhanced transcriptional activity mediated by the PR NTD, and deletion mutations showed that a region (amino acids 327–428), similar to that required for TBP-induced folding, was required for functional response. TBP also increased steroid receptor co-activator 1 (SRC-1) interaction with the PR NTD and cooperated with SRC-1 to stimulate NTD-dependent transcriptional activity. These data suggest that TBP can mediate structural reorganization of the NTD to facilitate the binding of co-activators required for maximal transcriptional activation. Background: The mechanism of action of the N-terminal domain (NTD) of the progesterone receptor is not well understood. Results: We show the PR NTD adopts a functional folded conformation by undergoing disorder-order transition via binding to a target protein, TBP. Conclusion: This structural reorganization of the NTD facilitates binding of co-activators required for transcriptional activation. Significance: A novel mechanism of PR-dependent transcriptional activation is defined.

Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17β-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.