Structure-Function Analysis of the Estrogen Receptor α Corepressor Scaffold Attachment Factor-B1

Abstract

Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ER␣)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable Cterminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ER␣ activity. SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ER␣ activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.SAFB1 1 belongs to a family of nuclear proteins that are localized in the nuclear matrix. The matrix is a proteinaceous structure consisting of a network of ribonucleoproteins and non-histone proteins, such as transcription factors, that serve as a scaffold for the higher organization of chromatin into loop structures (1). A characteristic of SAFB family members is the presence of an N-terminal SAF box (2-4) that binds to DNA regulatory regions termed scaffold/matrix attachment regions (S/MARs). S/MARs are bound to the nuclear matrix, define the loop structure of higher order chromatin (5-8), divide the genome into structural and functional domains, and are implicated in the regulation of gene expression (1).SAFB proteins contain a central RNA recognition motive (RRM), which suggests a role in mRNA processing. Because SAFB1 also has been shown to interact with members of the RNA processing machinery and with RNA polymerase II, it has been suggested that this protein is part of a "transcriptosome" complex, coupling chromatin structure to transcription and RNA processing (9 -12).We have previously reported that SAFB1 plays an important role in breast cancer because its overexpression results in growth inhibition (13). SAFB1 maps to a chromosomal locus that displays unusually high rates of loss of heterozygosity (14), and mutations have been identified in breast tumors (14). We have also shown that SAFB1 can bind to and repress transcriptional activity of the estrogen receptor ␣ (ER␣), thereby functioning as an ER␣ corepressor (15).ER␣ is a steroid receptor that regulates transcription of genes involved in proliferation, apoptosis, migration, and other cellular processes (16). ER␣ corepressors and coactivators are components of large protein complexes that tightly control the activity of ER␣ (17,18). Although the role of coactivators in the activity of ER␣ is well established, the role of ER␣ corepressors is less clear. Based on recent studies, it is believed that ER␣ corepr...

Attenuation of estrogen receptor α-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor

“…S4A, lanes 5,7). Of note, several NR coregulators have been shown to regulate both liganddependent and-independent transcription, including ER␣ corepressor SAFB1 (Townson et al 2004) and coactivator CoCoA .…”

The role of nuclear organization in cancer

“…SAFB1 and SAFB2 function as co-repressors of the ER to create a repressed chromatin structure through indirect recruitment of histone deacetylases and other regulatory factors, including cJun, PPARα, PPARβ, PPARγ and VDR [69,74,75]. Patients with ER-positive breast cancer have a better prognosis than those that are ER-negative, as they can be treated with oestrogen antagonists such as tamoxifen.…”

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…Conversely, weaker interactions may be maintained in the presence of low salt, but there may be a risk of artefactual interactions. [10], hnRNPC [10], hnRNPD [120], hnRNPG [25,84,121], hnRNPI [10], hnRNPK [10,122], hnRNPU [10] SR proteins SRSF1 [33], SRSF7 [26], SRSF9 [26,33], SREK1 [88], SRRM1 [123] SR protein kinase SRPK1 [89] Chromatin CHD1 [72], NCOR [37,71], HDAC3, [37,71] BRG1 [73], Matrin3 [54] Transcription RNAPolII [33], TAF II 68 [124], steroid receptors [67][68][69], P53 [70] Miscellaneous PIAS1 [37], ZO-2 [125], Zbed4 [126] Also, co-immunoprecipitation does not distinguish between direct protein interactions and interactions mediated by 'bridging' molecules, such as other proteins or nucleic acids. Formation of homo-and hetero-dimers of SAFB1 and SAFB2, as well as interactions with several hnRNP proteins, has been observed (see Table 1 for a summary of some of the proteins shown to interact with SAFB proteins).…”