Scaffold Attachment Factor B1 Research Articles

Abstract P6-11-01: Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer

Abstract Historically known as a tumor suppressor by estrogen receptor (ER) co-repression in breast cancer (BC), the matrix binding protein Scaffold Attachment Factor B1 (SAFB) binds scaffold or matrix attachment region DNA elements (S/MAR DNA) in eukaryotic DNA. SAFB1 plays a role in cellular stress response, DNA repair, differentiation, and apoptosis. SAFB loss in ER-independent BC and pancreatic cancer (PC) patients resulted in poor survival rates, hinting at the role of SAFB1 as a tumor suppressor. To understand the tumor suppressive mechanism of SAFB1, we performed shRNA-mediated knockdown (KD) of SAFB in PC cell lines and triple-negative breast cancer (TNBC) cells, an aggressive subgroup of BC. Analysis of onco-properties showed an increase in clonogenicity potential and cell proliferation in SAFB KDs in both PDACs and TNBCs cells. Further, RNA-Seq analysis of SAFB KDs in the TNBC cell line revealed activation of the mevalonate (MVA) pathway and the resulting cholesterol biosynthesis as the key metabolic change. Both pancreatic ductal adenocarcinoma (PDACs) and TNBC exhibited higher levels of MVA pathway gene expression upon loss of SAFB. Sterol regulatory element binding proteins (SREBP) 1 and 2 dictate cholesterol biosynthesis, and SREBP2 promotes tumor properties via the MVA pathway. Molecular and metabolic analysis of SAFB KD TNBC showed an increase in lipid droplets and SREBP2 maturation. Using Chromatin Immunoprecipitation and quantitative real-time PCR (ChIP-qPCR) in TNBC we demonstrate the direct binding of SAFB to the SREBP2 promoter region. In addition, byproducts of the MVA pathway have been shown to activate YAP/TAZ-dependent tissue homeostasis and tumorigenesis. We also observed increased YAP1 mRNA levels and decreased YAP1 (Ser127) phosphorylation with SAFB loss in TNBC, explaining the aggressive tumorigenicity gained with SAFB loss. Our study thus far suggests SAFB has overt control over the SREBP2-MVA-YAP1 pathway, and loss of SAFB results in an enhanced tumor phenotype. Citation Format: Meron Ghidey, Divya Murthy, Sukjin Yang, Songyeon Ahn, Jun Hyoung Park, Benny Kaipparettu. Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-01.

SENP7 deSUMOylase-governed transcriptional program coordinates sarcomere assembly and is targeted in muscle atrophy.

Disorganization of the basic contractile unit of muscle cells, i.e., the sarcomeres, leads to suboptimal forcegeneration and is a hallmark of muscle atrophy. Here, we demonstrate that the nuclear role of SENP7 deSUMOylase is pivotal for sarcomere organization. SENP7 expression is temporally upregulated in mature muscle cells and directly regulates transcription of the myosin heavy chain (MyHC-IId) gene. We identify SENP7-dependent deSUMOylation of flightless-1 (Fli-I) as a signal for Fli-I association with scaffold attachment factor b1 (Safb1). SENP7 deficiency leads to higher Fli-I SUMOylation and lower chromatin residency of Safb1, thus generating transcriptionally incompetent chromatin conformation on MyHC-IId. Consequently, lower expression of MyHC-IId causes sarcomere disorganization and disrupted muscle cell contraction. Remarkably, cachexia signaling impedes the SENP7-governed transcriptional program, leading to muscle atrophy, with profound loss of motor protein MyHC-IId. We propose a SENP7-driven distinct transcription program as paramount for muscle cell function, which was found targeted in cachexia.

Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1.

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, β, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRβ, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

Radiotherapy changes the salivary proteome in head and neck cancer patients: evaluation before, during, and after treatment.

Salivary glands are affected during radiotherapy in the head and neck region, leading to a reduction in salivary flow and changes its composition. Besides negatively affecting the oral soft tissues, this can also lead to dental impairment. Thus, we evaluated the effect of radiotherapy in the proteomic profile of the saliva in patients with head and neck cancer (HNC). HNC patients had their saliva collected before (BRT), during (2-5 weeks; DRT), and after (3-4 months; ART) radiotherapy. Saliva was also collected from healthy volunteers (control; C). Samples were processed for proteomic analysis. In total, 1055 proteins were identified, among which 46 were common to all groups, while 86, 86, 286, and 395 were exclusively found in C, BRT, DRT, and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and several cystatins. When compared with C, salivary proteins of BRT patients showed increases cystatin-C, lysozyme C, histatin-1, and proline-rich proteins CONCLUSION/CLINICAL REVELANCE: Both HNC and radiotherapy remarkably change the salivary protein composition. Altogether, our results, for the first time, suggest investigating alpha-enolase levels in saliva DRT in future studies as a possible biomarker and strategy to predict the efficiency of the treatment. Moreover, our data provide important insights for designing dental products that are more effective for these patients and contribute to a better understanding of the progressive changes in salivary proteins induced by radiotherapy. Graphical abstract.

Safb1 Regulates Cell Fate Determination in the Adult Brain by Enhancing mRNA Cleavage

During brain homeostasis, stem cell fate determination is crucial to guarantee function, adaptation and regeneration while preventing neurodegeneration and cognitive impairment. How neural stem cells (NSCs) are instructed to generate neurons or glia is not well understood. Here we addressed how fate is resolved in multipotent adult hippocampal NSCs, and identify Scaffold Attachment Factor B1 (Safb1) as a determinant of neuron production by blocking glial commitment. Safb1 is sufficient to block oligodendrocytic differentiation of NSCs by preventing expression of the transcription factor NFIB at the post-transcriptional level. Detailed interrogation of the Drosha interactome and functional validation revealed that Safb1 enhances NFIB mRNA cleavage in a Drosha-dependent fashion. Thus, our study provides a cellular mechanism for selective NSC fate regulation by post-transcriptional destabilization of mRNAs. Given the importance of NSC maintenance and fate determination in the adult brain, our findings have major implications for cell-specific gene expression, brain disease and aging.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.

FUS interacts with nuclear matrix-associated protein SAFB1 as well as Matrin3 to regulate splicing and ligand-mediated transcription.

FUS (Fused-in-Sarcoma) is a multifunctional DNA/RNA binding protein linked to familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Since FUS is localized mainly in the nucleus with nucleo-cytoplasmic shuttling, it is critical to understand physiological functions in the nucleus to clarify pathogenesis. Here we report a yeast two-hybrid screening identified FUS interaction with nuclear matrix-associated protein SAFB1 (scaffold attachment factor B1). FUS and SAFB1, abundant in chromatin-bound fraction, interact in a DNA-dependent manner. N-terminal SAP domain of SAFB1, a DNA-binding motif, was required for its localization to chromatin-bound fraction and splicing regulation. In addition, depletion of SAFB1 reduced FUS’s localization to chromatin-bound fraction and splicing activity, suggesting SAFB1 could tether FUS to chromatin compartment thorough N-terminal DNA-binding motif. FUS and SAFB1 also interact with Androgen Receptor (AR) regulating ligand-dependent transcription. Moreover, FUS interacts with another nuclear matrix-associated protein Matrin3, which is muted in a subset of familial ALS cases and reportedly interacts with TDP-43. Interestingly, ectopic ALS-linked FUS mutant sequestered endogenous Matrin3 and SAFB1 in the cytoplasmic aggregates. These findings indicate SAFB1 could be a FUS’s functional platform in chromatin compartment to regulate RNA splicing and ligand-dependent transcription and shed light on the etiological significance of nuclear matrix-associated proteins in ALS pathogenesis.

Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells.

Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3′ or 5′ untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.

Scaffold attachment factor B1: an intrinsic inhibitor of androgen receptor downregulated in prostate cancer

The androgen receptor (AR) is a pleiotrophic transcription factor that regulates expression of a large number of genes involved in many diverse cellular processes. The AR activation pathways have been studied extensively. However, the molecular mechanism and biological significance of AR inhibitory signals remain poorly understood. Mukhopadhyay et al. have now identified the nuclear matrix protein scaffold attachment factor B1 (SAFB1) as a novel AR corepressor. The authors found that SAFB1 physically associates with AR protein and inhibits AR transcriptional activity and androgen-sensitive gene expression. SAFB1 had no effect on chromatin occupancy of AR, but silencing of SAFB1 abolished recruitment MST1, a known AR repressor, at AR target loci. They also showed that SAFB1 interacts with EZH2, SUZ12 and EED, three core components of the Polycomb repressive complex 2 (PRC2), which catalyzes the gene repression histone modification H3 lysine 27 trimethylation (H3K27me3). The authors further showed that forced expression of SAFB1 increases H3K27me3 at AR target loci and this effect requires EZH2. Finally, the authors demonstrated that expression of SAFB1 is downregulated in human prostate cancer (PCa) specimens and SAFB1 knockdown results in an aggressive phenotype of PCa. These findings identify SAFB1 as an important node for integration of multiple inhibitory signals of AR, which represents a viable pathway for therapeutic intervention of PCa.

Scaffold attachment factor B1 regulates the androgen receptor in concert with the growth inhibitory kinase MST1 and the methyltransferase EZH2

The androgen receptor (AR) is a transcription factor that employs many diverse interactions with coregulatory proteins in normal physiology and in prostate cancer (PCa). The AR mediates cellular responses in association with chromatin complexes and kinase cascades. Here we report that the nuclear matrix protein, scaffold attachment factor B1 (SAFB1), regulates AR activity and AR levels in a manner that suggests its involvement in PCa. SAFB1 mRNA expression was lower in PCa in comparison with normal prostate tissue in a majority of publicly available RNA expression data sets. SAFB1 protein levels were also reduced with disease progression in a cohort of human PCa that included metastatic tumors. SAFB1 bound to AR and was phosphorylated by the MST1 (Hippo homolog) serine-threonine kinase, previously shown to be an AR repressor, and MST1 localization to AR-dependent promoters was inhibited by SAFB1 depletion. Knockdown of SAFB1 in androgen-dependent LNCaP PCa cells increased AR and prostate-specific antigen (PSA) levels, stimulated growth of cultured cells and subcutaneous xenografts and promoted a more aggressive phenotype, consistent with a repressive AR regulatory function. SAFB1 formed a complex with the histone methyltransferase EZH2 at AR-interacting chromatin sites in association with other polycomb repressive complex 2 (PRC2) proteins. We conclude that SAFB1 acts as a novel AR co-regulator at gene loci where signals from the MST1/Hippo and EZH2 pathways converge.

The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state

The regulation of skeletal muscle gene expression during myogenesis is mediated by lineage-specific transcription factors in combination with numerous cofactors, many of which modify chromatin structure. However, the involvement of scaffolding proteins that organize chromatin and chromatin-associated regulatory proteins has not extensively been explored in myogenic differentiation. Here, we report that Scaffold attachment factor b1 (Safb1), primarily associated with transcriptional repression, functions as a positive regulator of myogenic differentiation. Knockdown of Safb1 inhibited skeletal muscle marker gene expression and differentiation in cultured C2C12 myoblasts. In contrast, over-expression resulted in the premature expression of critical muscle structural proteins and formation of enlarged thickened myotubes. Safb1 co-immunoprecipitated with MyoD and was co-localized on myogenic promoters. Upon Safb1 knockdown, the repressive H3K27me3 histone mark and binding of the Polycomb histone methyltransferase Ezh2 persisted at differentiation-dependent gene promoters. In contrast, the appearance of histone marks and regulators associated with myogenic gene activation, such as myogenin and the SWI/SNF chromatin remodelling enzyme ATPase, Brg1, was blocked. These results indicate that the scaffold protein Safb1 contributes to the activation of skeletal muscle gene expression during myogenic differentiation by facilitating the transition of promoter sequences from a repressive chromatin structure to one that is transcriptionally permissive.

SAFB1- and SAFB2-mediated transcriptional repression: relevance to cancer

SAFB1 (scaffold attachment factor B1) and a second family member SAFB2, are multifunctional proteins implicated in a variety of cellular processes including cell growth, apoptosis and stress response. Their potential function as tumour suppressors has been proposed based on well-described roles in tran-scriptional repression. The present review summarizes the current knowledge of SAFB1 and SAFB2 proteins in transcriptional repression with relevance to cancer.

Scaffold attachment factor B (SAFB)1 and SAFB2 cooperatively inhibit the intranuclear mobility and function of ERα

Estrogen receptor alpha (ERα) plays a key role in physiological and pathophysiological processes as a ligand-activated transcriptional factor that is regulated by cofactors. ERα-mediated transcriptional regulation is closely correlated with the mobility of ERα in the nucleus in association with the nuclear matrix, the framework for nuclear events including transcription. However, the relationship between ERα mobility and the cofactors of ERα is unclear. Scaffold attachment factor B1 (SAFB1) and its paralog SAFB2 are nuclear matrix binding proteins that have been characterized as ERα corepressors. Here, using chimeric fluorescent proteins (FPs), we show that SAFB1 and SAFB2 colocalize with ERα in the nucleus of living cells after 17β-estradiol (E2) treatment. Co-immunoprecipitation (co-IP) experiments indicated that ERα interacts with both SAFB1 and SAFB2 in the presence of E2. Fluorescence recovery after photobleaching analysis revealed that SAFB1 and SAFB2 each decrease ERα mobility, and interestingly, coexpression of SAFB1 and SAFB2 causes a synergistic reduction in ERα dynamics under E2 treatment. In accordance with these mobility changes, ERα-mediated transcription and proliferation are cooperatively inhibited by SAFB1 and SAFB2. These results indicate that SAFB1 and SAFB2 are crucial repressors for ERα dynamics in association with the nuclear matrix and that their synergistic regulation of ERα mobility is sufficient for inhibiting ERα function.

Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.

Co-repressor activity of scaffold attachment factor B1 requires sumoylation

Sumoylation is an emerging modification associated with a variety of cellular processes including the regulation of transcriptional activities of nuclear receptors and their coregulators. As SUMO modifications are often associated with transcriptional repression, we examined if sumoylation was involved in modulation of the transcriptional repressive activity of scaffold attachment factor B1. Here we show that SAFB1 is modified by both the SUMO1 and SUMO2/3 family of proteins, on lysine’s K231 and K294. Further, we demonstrate that SAFB1 can interact with PIAS1, a SUMO E3 ligase which mediates SAFB1 sumoylation. Additionally, SENP1 was identified as the enzyme desumoylating SAFB1. Mutation of the SAFB1 sumoylation sites lead to a loss of transcriptional repression, at least in part due to decreased interaction with HDAC3, a known transcriptional repressor and SAFB1 binding partner. In summary, the transcriptional repressor SAFB1 is modified by both SUMO1 and SUMO2/3, and this modification is necessary for its full repressive activity.

The Werner syndrome helicase protein is required for cell proliferation, immortalization, and tumorigenesis in Scaffold Attachment Factor B1 deficient mice

Werner syndrome (WS) is a rare disorder characterized by the premature onset of several pathologies associated with aging. The gene responsible for WS codes for a RecQ-type DNA helicase and is believed to be involved in different aspects of DNA repair, replication, and transcription. We recently identified the Scaffold attachment factor B1 (SAFB1) as a potential interactants in human cells. SAFB1 is a multifunctional protein that binds both nucleic acids and is involved in the attachment of chromatin to the nuclear matrix, transcription, and stress response. Mice lacking SAFB1 exhibit developmental abnormalities in their lungs, high incidence of perinatal lethality, and adults develop different types of tumors. Mouse embryonic fibroblasts from Safb1-null animals are immortalized in culture. In this study, mice with a mutation in the helicase domain of the Wrn gene were crossed to Safb1-null mice. Double homozygous mutant mice exhibited increased apoptosis, a lower cell proliferation rate in their lungs and a higher incidence of perinatal death compared to Safb1-null mice. Few double homozygous mutants survived weaning and died before the age of six months. Finally, mouse embryonic fibroblasts lacking a functional Wrn helicase inhibited the immortalization of Safb1-null cells. These results indicate that an intact Wrn protein is required for immortalization and tumorigenesis in Safb1-null mice.

SAFB1 interacts with and suppresses the transcriptional activity of p53

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity. Structured summaryp53physically interacts with SRPK1a: shown by two hybrid (view interaction)p53physically interacts with SRPK1a: shown by pull down (view interaction)p53physically interacts with SRPK1a: shown by anti bait coimmunoprecipitation (view interaction)p53physically interacts with SRPK1a: shown by anti tag coimmunoprecipitation (view interaction)SAFB1physically interacts with p53: shown by pull down (view interactions 1, 2)SAFB1physically interacts with p53: shown by anti bait coimmunoprecipitation (view interactions 1, 2)SAFB1 and p53colocalize: shown by fluorescence microscopy (view interaction)SAFB2physically interacts with p53: shown by pull down (view interaction)

Analysis of SAFB1 and SAFB2 Knockout Mice Reveals Non-Redundant Functions of the Two Estrogen Receptor Co-Repressor Paralogs.

Abstract The Scaffold Attachment Factor B1 (SAFB1) and B2 (SAFB2) have been shown to be involved in chromatin organization, transcriptional regulation, and RNA processing. The paralogs SAFB1 and SAFB2 share 74% similarity at the amino acid level, with up to 98% similarity in some functional domains. These functional domains include DNA and RNA-binding domains, and a C-terminal repression domain. We have previously shown that SAFB1 and SAFB2 function as estrogen receptor (ER) corepressors – they directly bind to ER, and repress its transcriptional activity. There is also evidence that both proteins play a role in breast cancer; their shared chromosomal locus on chromosome 19p13 displays high rates of LOH, and protein loss was associated with worse survival of breast cancer patients.To understand the roles of SAFB1 and SAFB2 in development and function of hormone responsive tissues, we generated gene-specific knockout (KO) mouse models. Deletion of SAFB1 resulted in a high degree of embryonic and perinatal lethality. Surviving SAFB1 KO mice displayed severe growth retardation associated with low serum IGF-I levels, male infertility and female subfertility. In contrast, SAFB2 KO mice were born at the expected Mendelian ratio and did not show any obvious defects in growth or fertility. Initial gross pathology analysis has not yet revealed any significant defects.Since the SAFB proteins are known to play a role in estrogen response, we analyzed mammary gland development in the two mouse models. Young virgin SAFB1 KO mice showed delayed mammary gland development with a significantly reduced number of terminal end buds and decreased outgrowth compared to wild type (WT) controls. This was likely a result of delayed puberty, due to low IGF-I levels, since at the four month time point, mammary gland growth was restored in the virgin SAFB1 KO mice to that seen in WT controls. Interestingly, the KO glands exhibited increased alveolar development and side branching. To measure proliferation directly, we performed mammary gland transplantation experiments, thereby excluding secondary effects due to systemic defects. These studies showed a significant increase in proliferation in SAFB1 KO glands compared to WT control glands. In contrast to the SAFB1-null mice, we did not observe any obvious mammary gland defects in pubertal or adult SAFB2-null mice. However, mammary glands from aged virgin SAFB2 mice (1.5 years old) showed extensive side branching and precocious development of alveolar buds resembling glands of late pregnant mice. This was associated with increased proliferation of alveolar cells in the SAFB2 KO glands.In summary, genetic ablation of the ER co-repressors SAFB1 and SAFB2 results in defects in mammary gland development. In general, the two mouse models have very different phenotypes, revealing diverse and non-redundant functions of SAFB1 and SAFB2, findings that were unexpected based on their high sequence similarity. We are currently performing additional studies to finalize characterization of the in vivo phenotypes, focusing on hormone responsive tissues, and also to understand the mechanism underlying the observed phenotypes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2162.

SAFB1's multiple functions in biological control—lots still to be done!

The examination of scaffold attachment factor B1 (SAFB1) and its multiple functions and tasks in cellular processes provides insight into its role in diseases, such as cancer. SAFB1 is a large multi-domain protein with well-described functions in transcriptional repression, and RNA splicing. It is ubiquitously expressed, and has been shown to be important in numerous cellular processes including cell growth, stress response, and apoptosis. SAFB1 is part of a protein family with at least two other family members, SAFB2 and the SAFB-like transcriptional modulator SLTM. The goal of this prospect article is to summarize known functions of SAFB1, and its roles in cellular processes, but also to speculate on less well described, novel attributes of SAFB1, such as a potential role in chromatin organization. This timely review shows aspects of SAFB1, which are proving to have a complexity far greater than was previously thought.

The enzymatic activity of SR protein kinases 1 and 1a is negatively affected by interaction with scaffold attachment factors B1 and 2

SR protein kinases (SRPKs) phosphorylate Ser/Arg dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. Phosphorylation by SRPKs constitutes a major way of regulating such cellular mechanisms. In the past, we have shown that SRPK1a interacts with the nuclear matrix protein scaffold attachment factor B1 (SAFB1) via its unique N-terminal domain, which differentiates it from SRPK1. In this study, we show that SAFB1 inhibits the activity of both SRPK1a and SRPK1 in vitro and that its RE-rich region is redundant for the observed inhibition. We demonstrate that kinase activity inhibition is caused by direct binding of SAFB1 to SRPK1a and SRPK1, and we also present evidence for the in vitro binding of SAFB2 to the two kinases, albeit with different affinity. Moreover, we show that both SR protein kinases can form complexes with both scaffold attachment factors B in living cells and that this interaction is capable of inhibiting their activity, depending on the tenacity of the complex formed. Finally, we present data demonstrating that SRPK/SAFB complexes are present in the nucleus of HeLa cells and that the enzymatic activity of the nuclear matrixlocalized SRPK1 is repressed. These results suggest a new role for SAFB proteins as regulators of SRPK activity and underline the importance of the assembly of transient intranuclear complexes in cellular regulation.