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Human estrogen receptor alpha (hERα) is a hormone-responsive nuclear receptor (NR) involved in cell growth and survival that contains both a DNA-binding domain (DBD) and a ligand-binding domain (LBD). Functionally relevant inter-domain interactions between the DBD and LBD have been observed in several other NRs, but for hERα, the detailed structural architecture of the complex is unknown. By utilizing integrated complementary techniques of small-angle X-ray scattering, hydroxyl radical protein footprinting and computational modeling, here we report an asymmetric L-shaped “boot” structure of the multidomain hERα and identify the specific sites on each domain at the domain interface involved in DBD–LBD interactions. We demonstrate the functional role of the proposed DBD–LBD domain interface through site-specific mutagenesis altering the hERα interfacial structure and allosteric signaling. The L-shaped structure of hERα is a distinctive DBD–LBD organization of NR complexes and more importantly, reveals a signaling mechanism mediated by inter-domain crosstalk that regulates this receptor’s allosteric function., The human estrogen receptor alpha (hERα) is a hormone-responsive transcription factor. Here the authors combine small-angle X-ray scattering, hydroxyl radical protein footprinting and computational modeling and show that multidomain hERα adopts an L-shaped boot-like architecture revealing a cross-talk between its DNA-binding domain and Ligand-binding domain.

Glucocorticoid receptor (GC), a founding member of the nuclear hormone receptor superfamily, is a glucocorticoid-activated transcription factor that regulates gene expression and controls the development and homeostasis of human podocytes. Synthetic glucocorticoids are the standard treatment regimens for proteinuria (protein in the urine) and nephrotic syndrome (NS) caused by kidney diseases. These include minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and immunoglobulin A nephropathy (IgAN) or subsequent complications due to diabetes mellitus or HIV infection. However, unwanted side effects and steroid-resistance remain major issues for their long-term use. Furthermore, the mechanism by which glucocorticoids elicit their renoprotective activity in podocyte and glomeruli is poorly understood. Podocytes are highly differentiated epithelial cells that contribute to the integrity of kidney glomerular filtration barrier. Injury or loss of podocytes leads to proteinuria and nephrotic syndrome. Recent studies in multiple experimental models have begun to explore the mechanism of GC action in podocytes. This review will discuss progress in our understanding of the role of glucocorticoid receptor and glucocorticoids in podocyte physiology and their renoprotective activity in nephrotic syndrome.

IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.

Oral cancer, a subtype of head and neck cancer, is characterized by increased infiltrating regulatory T cells (Treg); however, the pathological significance of the increase in Tregs in disease prognosis and progression and their underlying mechanism remain unestablished. C‐C motif chemokine ligand 22 (CCL22) has been implicated in the recruitment of Tregs. We used RT‐qPCR to determine CCL22 mRNA expression in clinical specimens and cultured cells. Loss‐of‐function and gain‐of‐function studies were carried out to analyze the effects of CCL22 modulations on cell proliferation, migration, invasion, and tumorigenesis and the mechanism involved in the deregulation of CCL22. In oral cancer specimens, CCL22 mRNA was upregulated. The increase was not only associated with reduced disease‐free survival but also strongly correlated with an increase in FOXP3 mRNA, a master regulator of Treg development and functions. Silencing CCL22 expression reduced cell proliferation, migration, and invasion, whereas ectopic overexpression showed opposite effects. Manipulation of CCL22 expression in cancer cells altered tumorigenesis in both immune‐compromised and ‐competent mice, supporting both autonomous and non‐autonomous actions of CCL22. Release of interleukin 1β (IL‐1β) from cancer‐associated fibroblasts (CAF) induces CCL22 mRNA expression in oral cancer cells by activating transcription factor nuclear factor kappa B (NF‐κB). Our data support a model in which CAF‐derived IL‐1β, CCL22, and its receptor CCR4 foster a protumor environment by promoting cell transformation and Treg infiltration. Intervention of the IL‐1β‐CCL22‐CCR4 signaling axis may offer a novel therapeutic strategy for oral cancer treatment.

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.

The S100A2 protein is an important regulator of keratinocyte differentiation, but its role in wound healing remains unknown. We establish epithelial-specific S100A2 transgenic (TG) mice and study its role in wound repair using punch biopsy wounding assays. In line with the observed increase in proliferation and migration of S100A2-depleted human keratinocytes, mice expressing human S100A2 exhibit delayed cutaneous wound repair. This was accompanied by the reduction of re-epithelialization as well as a slow, attenuated response of Mcp1, Il6, Il1β, Cox2, and Tnf mRNA expression in the early phase. We also observed delayed Vegfa mRNA induction, a delayed enhancement of the Tgfβ1-mediated alpha smooth muscle actin (α-Sma) axis and a differential expression of collagen type 1 and 3. The stress-activated p53 tumor suppressor protein plays an important role in cutaneous wound healing and is an S100A2 inducer. Notably, S100A2 complexes with p53, potentiates p53-mediated transcription and increases p53 expression both transcriptionally and posttranscriptionally. Consistent with a role of p53 in repressing NF-κB-mediated transcriptional activation, S100A2 enhanced p53-mediated promoter suppression of Cox2, an early inducible NF-κB target gene upon wound injury. Our study thus supports a model in which the p53-S100A2 positive feedback loop regulates wound repair process.

Promyelocytic leukemia protein (PML) was originally identified as a fusion partner of retinoic acid receptor alpha in acute promyelocytic leukemia patients with the (15;17) chromosomal translocation, giving rise to PML–RARα and RARα–PML fusion proteins. A body of evidence indicated that PML possesses tumor suppressing activity by regulating apoptosis, cell cycle, senescence and DNA damage responses. PML is enriched in discrete nuclear substructures in mammalian cells with 0.2–1 μm diameter in size, referred to as alternately Kremer bodies, nuclear domain 10, PML oncogenic domains or PML nuclear bodies (NBs). Dysregulation of PML NB formation results in altered transcriptional regulation, protein modification, apoptosis and cellular senescence. In addition to PML NBs, PML is also present in nucleoplasm and cytoplasmic compartments, including the endoplasmic reticulum and mitochondria-associated membranes. The role of PML in tumor suppression has been extensively studied but increasing evidence indicates that PML also plays versatile roles in stem cell renewal, metabolism, inflammatory responses, neural function, mammary development and angiogenesis. In this review, we will briefly describe the known PML regulation and function and include new findings.

Glomerular podocytes are highly specialized terminally differentiated cells that act as a filtration barrier in the kidney. Mutations in the actin-binding protein, α-actinin 4 (ACTN4), are linked to focal segmental glomerulosclerosis (FSGS), a chronic kidney disease characterized by proteinuria. Aberrant activation of NF-κB pathway in podocytes is implicated in glomerular diseases including proteinuria. We demonstrate here that stable knockdown of ACTN4 in podocytes significantly reduces TNFα-mediated induction of NF-κB target genes, including IL-1β and NPHS1, and activation of an NF-κB-driven reporter without interfering with p65 nuclear translocation. Overexpression of ACTN4 and an actin binding-defective variant increases the reporter activity. In contrast, an FSGS-linked ACTN4 mutant, K255E, which has increased actin binding activity and is predominantly cytoplasmic, fails to potentiate NF-κB activity. Mechanistically, IκBα blocks the association of ACTN4 and p65 in the cytosol. In response to TNFα, both NF-κB subunits p65 and p50 translocate to the nucleus, where they bind and recruit ACTN4 to their targeted promoters, IL-1β and IL-8. Taken together, our data identify ACTN4 as a novel coactivator for NF-κB transcription factors in podocytes. Importantly, this nuclear function of ACTN4 is independent of its actin binding activity in the cytoplasm.

The actin crosslinking protein α-actinin-4 (ACTN4) is emerging as an important contributor to the pathogenesis of cancer. This has largely been attributed to its role in regulating cytoskeleton organization and its involvement in transcriptional regulation of gene expression. Here we report a novel function of ACTN4 as a scaffold necessary for stabilization of receptor-interacting protein kinase 1 (RIPK1) that we have recently found to be an oncogenic driver in melanoma. ACTN4 bound to RIPK1 and cellular inhibitor of apoptosis protein 1 (cIAP1) with its actin-binding domain at the N-terminus and the CaM-like domain at the C-terminus, respectively. This facilitated the physical association between RIPK1 and cIAP1 and was critical for stabilization of RIPK1 that in turn activated NF-κB. Functional investigations showed that silencing of ACTN4 suppressed melanoma cell proliferation and retarded melanoma xenograft growth. In contrast, overexpression of ACTN4 promoted melanocyte and melanoma cell proliferation and moreover, prompted melanocyte anchorage-independent growth. Of note, the expression of ACTN4 was transcriptionally activated by NF-κB. Taken together, our findings identify ACTN4 as an oncogenic regulator through driving a feedforward signaling axis of ACTN4-RIPK1-NF-κB, with potential implications for targeting ACTN4 in the treatment of melanoma.

PML plays a critical role in the maintenance of ROS homeostasis via a unique mechanism in which PML functions as an oxidative sensor to regulate the expression of antioxidant genes through Nrf2. PML is also indispensable for sulforaphane-mediated ROS generation, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis., Oxidative stress is a consequence of an imbalance between reactive oxygen species (ROS) production and the ability of the cytoprotective system to detoxify the reactive intermediates. The tumor suppressor promyelocytic leukemia protein (PML) functions as a stress sensor. Loss of PML results in impaired mitochondrial complex II activity, increased ROS, and subsequent activation of nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidative pathway. We also demonstrate that sulforaphane (SFN), an antioxidant, regulates Nrf2 activity by controlling abundance and subcellular distribution of PML and that PML is essential for SFN-mediated ROS increase, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis. Taking the results together, we have uncovered a novel antioxidative mechanism by which PML regulates cellular oxidant homeostasis by controlling complex II integrity and Nrf2 activity and identified PML as an indispensable mediator of SFN activity.

The promyelocytic leukemia protein is a well known tumor suppressor, but its role in metabolism is largely unknown. Mice with a deletion in the gene for PML (KO mice) exhibit altered gene expression in liver, adipose tissue, and skeletal muscle, an accelerated rate of fatty acid metabolism, abnormal glucose metabolism, constitutive AMP-activating kinase (AMPK) activation, and insulin resistance in skeletal muscle. Last, an increased rate of energy expenditure protects PML KO mice from the effects of obesity induced by a Western diet. Collectively, our study uncovers a previously unappreciated role of PML in the regulation of metabolism and energy balance in mice.

Nucleocytoplasmic shuttling of class IIa of histone deacetylases (HDACs) is a key mechanism that controls cell fate and animal development. We have identified the filamin B (FLNB) as a novel HDAC7-interacting protein that is required for temporal and spatial regulation of vascular endothelial growth factor (VEGF)-mediated HDAC7 cytoplasmic sequestration. This interaction occurs in the cytoplasm and requires monoubiquitination of an evolutionarily conserved lysine 1147 (K1147) in the immunoglobulin (Ig)-like repeat 10 (R10) of FLNB and the nuclear localization sequence of HDAC7. Inhibition of protein kinase C (PKC) blocks VEGF-induced ubiquitination of FLNB and its interaction with HDAC7. Small interfering RNA (siRNA) knockdown of FLNB or ubiquitin (Ub) in human primary endothelial cells blocks VEGF-mediated cytoplasmic accumulation of HDAC7, reduces VEGF-induced expression of the HDAC7 target genes Mmp-10 and Nur77, and inhibits VEGF-induced vascular permeability. Using dominant negative mutants and rescue experiments, we demonstrate the functional significance of FLNB K1147 to interfere with the ability of phorbol myristate acetate (PMA) to promote FLNB-mediated cytoplasmic accumulation of HDAC7. Taken together, our data show that VEGF and PKC promote degradation-independent protein ubiquitination of FLNB to control intracellular trafficking of HDAC7.

Glucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that α actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-κB (p65) binding sites on GR-transrepressed promoters such as IL-1β, IL-6, and IL-8. Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.

Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Muller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.

Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor α (TNFα) and interferon α (IFNα) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNFα- and IFNα-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNFα- and IFNα-induced PML accumulation and relieves TNFα- and IFNα-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin β1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNFα- and IFNα-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration.

Mutations in α-actinin 4 (ACTN4) are linked to familial forms of focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria due to podocyte injury. The mechanisms underlying ACTN4 mutant-associated FSGS are not completely understood. Although α-actinins are better known to cross-link actin filaments and modulate cytoskeletal organization, we have previously shown that ACTN4 interacts with transcription factors including estrogen receptor and MEF2s and potentiates their transcriptional activity. Nuclear receptors including retinoic acid receptor (RAR) have been proposed to play a protective role in podocytes. We show here that ACTN4 interacts with and enhances transcriptional activation by RARα. In addition, FSGS-linked ACTN4 mutants not only mislocalized to the cytoplasm, but also lost their ability to associate with nuclear receptors. Consequently, FSGS-linked ACTN4 mutants failed to potentiate transcriptional activation by nuclear hormone receptors in podocytes. In addition, overexpression of these mutants suppressed the transcriptional activity mediated by endogenous wild-type ACTN4 possibly by a cytoplasmic sequestration mechanism. Our data provide the first link between FSGS-linked ACTN4 mutants and transcriptional activation by nuclear receptor such as RARα and peroxisome proliferator-activated receptor γ.

The promyelocytic leukemia (PML) protein is a tumor suppressor that has an important role in several cellular processes, including apoptosis, viral infection, DNA damage repair, cell cycle regulation, and senescence. PML is an essential component of sub-nuclear structures called PML nuclear bodies (NBs). Our laboratory has previously demonstrated that the peptidyl-prolyl cis-trans isomerase, Pin1, binds and targets PML for degradation in a phosphorylation-dependent manner. To further elucidate the mechanisms underlying Pin1-mediated PML degradation, we aimed to identify one or more factors that promote PML phosphorylation. Here we show that treatment with U0126, an inhibitor of the ERK2 upstream kinases MEK1/2, leads to an increase in PML protein accumulation and an inhibition of the interaction between Pin1 and PML in MDA-MB-231 breast cancer cells. Consistent with this observation, phosphorylated ERK2 partially co-localized with PML NBs. Although U0126 up-regulated exogenous wild-type PML levels, it did not have an effect on the steady-state level of a mutant form of PML that is defective in binding Pin1. In addition, exogenous wild-type, but not Pin1 binding-defective PML protein expression levels were decreased by overexpression of ERK2. In contrast, knockdown of ERK2 by siRNA resulted in an increase in PML protein levels and an increase in the formation of PML NBs. Using phospho-specific antibodies, we identified Ser-403 and Ser-505 as the ERK2 targets that promote Pin1-mediated PML degradation. Finally, we demonstrated that EGF induced activation of ERK and interaction between PML and phosphorylated ERK resulting in a decrease in PML protein levels. Taken together, our results support a model in which Pin1 promotes PML degradation in an ERK2-dependent manner.

Promyelocytic leukemia protein (PML) was originally identified as part of a chromosomal translocation that contributes to the development of acute promyelocytic leukemia (APL). Since its discovery, PML has been found to play diverse roles in different cellular processes. Notably, PML has anti-proliferative and pro-apoptotic activity that supports its role as a tumor suppressor. We have previously shown that the peptidyl-prolyl isomerase Pin1 is able to affect cell proliferation and hydrogen peroxide (H(2)O(2))-mediated cell death through modulation of the steady-state levels of PML. We have extended these studies to show that the interaction between PML and Pin1 is targeted by multiple extracellular signals in the cell. We show that H(2)O(2) up-regulates and IGF-1 down-regulates PML expression in a Pin1-dependent manner. Interestingly, we found that H(2)O(2)- and IGF-1-mediated alteration in PML accumulation regulate MDA-MB-231 cell migration. Furthermore, we show that the control of cell migration by PML, and thus H(2)O(2) and IGF-1, results from PML-dependent decreased expression of integrin beta1 (ITGB1). Knockdown of Pin1 leads to decreased cell migration, lower levels of ITGB1 expression and resistance to IGF-1- and H(2)O(2)-induced changes in cell migration and ITGB1 expression. Taken together, our work identifies PML as a common target for H(2)O(2) and IGF-1 and supports a novel tumor suppressive role for PML in controlling cell migration through the expression of ITGB1.

The promyelocytic leukemia protein (PML) is involved in many cellular processes including cell cycle progression, DNA damage response, transcriptional regulation, viral infection, and apoptosis. These cellular activities often rely on the localization of PML to unique subnuclear structures known as PML nuclear bodies (NBs). More than 50 cellular proteins are known to traffic in and out of PML NBs, either transiently or constitutively. In order to understand the dynamics of these NBs, it is important to delineate the regulation of PML itself. PML is subject to extensive regulation at transcriptional, post-transcriptional, and post-translational levels. Many of these modes of regulation depend on the cellular context and the presence of extracellular signals. This review focuses on the current knowledge of regulation of PML under normal cellular conditions as well as the role for regulation of PML in viral infection and cancer.

Silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) is a transcriptional corepressor that participates in diverse signaling pathways and human diseases. However, regulation of SMRT stability remains largely unexplored. We show that the peptidyl-prolyl isomerase Pin1 interacts with SMRT both in vitro and in mammalian cells. This interaction requires the WW domain of Pin1 and SMRT phosphorylation. Pin1 regulates SMRT protein stability, thereby affecting SMRT-dependent transcriptional repression. SMRT phosphorylation at multiple sites is required for Pin1 interaction, and these sites can be phosphorylated by Cdk2, which interacts with SMRT. Cdk2-mediated phosphorylation of SMRT is required for Pin1 binding and decreases SMRT stability, whereas mutation of these phosphorylation sites abrogates Pin1 binding and stabilizes SMRT. Finally, decreases in SMRT stability occur in response to the activation of Her2/Neu/ErbB2, and this receptor functions upstream of both Pin1 and Cdk2 in the signaling cascade that regulates SMRT stability and cellular response to tamoxifen.

The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells.

Promyelocytic leukemia protein (PML) sumoylation has been proposed to control the formation of PML nuclear bodies (NBs) and is crucial for PML-dependent cellular processes, including apoptosis and transcriptional regulation. However, the regulatory mechanisms of PML sumoylation and its specific roles in the formation of PML NBs remain largely unknown. Here, we show that histone deacetylase 7 (HDAC7) knockdown reduces the size and the number of the PML NBs in human umbilical vein endothelial cells (HUVECs). HDAC7 coexpression stimulates PML sumoylation independent of its HDAC activity. Furthermore, HDAC7 associates with the E2 SUMO ligase, Ubc9, and stimulates PML sumoylation in vitro, suggesting that it possesses a SUMO E3 ligase-like activity to promote PML sumoylation. Importantly, HDAC7 knockdown inhibits tumor necrosis factor alpha-induced PML sumoylation and the formation of PML NBs in HUVECs. These results demonstrate a novel function of HDAC7 and provide a regulatory mechanism of PML sumoylation.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-α treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-α promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-α-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.

The alpha actinin (ACTN) family proteins are actin-bundling proteins that are evolutionarily conserved and possess several functional domains. Vertebrates including humans express four unique actinin genes that display distinct expression patterns and biochemical properties. Mutations in each of these actinins are associated with tissues-specific abnormalities. ACTN1 and ACTN4 are ubiquitously expressed and their major spliced isoforms harbor calcium-sensitive EF hands, while the expression of ACTN2 and ACTN3 are primarily restricted to muscle and are calcium-insensitive. Due to their ability to bind filamentous actin, ACTNs have been shown to regulate cytokinesis, cell adhesion, spreading, migration and signaling. In addition, a fraction of ACTN4 is localized in the nucleus where it interacts with transcription factors, histone modifying enzymes and chromatin remodeling proteins in order to stimulate transcription [1–3]. Notably, all ACTNs harbor an evolutionarily conserved motif,—LXXLL—(L, leucine, X, any amino acids), which is required for interaction with the nuclear receptor family of transcription factors [3]. Indeed, ACTN1 and ACTN2 have been shown to enhance nuclear receptor-mediated transcription [1, 4]. Thus, ACTNs join the list of several other cytoskeletal proteins that are capable of shuttling between the cytoplasm and the nucleus to control cell function [5]. In this thematic review series, we feature recent progress in our understanding of the biological and the pathological roles of the ACTNs and genetic lesions in this protein family that are associated with human diseases. Murphy and Young [6] first summarize genetic studies of actinin in invertebrates and cast an evolutionary perspective of this unique actin-binding protein family. They detail the similarities and differences among the 4 vertebrate ACTNs and provide an insightful discussion of actinin-related genetic disorders with primary focus on ACTN1, 2 and 3. For example, mutations in ACTN1 are associated with dominantly-inherited congenital macrothrombocytopenia (CMPT), a rare blood disorder, while mutations in ACTN2 are linked to hypertrophic cardiomyopathy (HCM). Interestingly, disease-associated ACTN1 and ACTN2 mutations are only found in the actin-binding domain, emphasizing the physiological significance of this functional domain. Surprisingly, ACTN3 is not essential and individuals with ACTN3 null alleles, due to a homozygous premature stop codon, are found in 16 % of world population. Interestingly, wild-type allele is found to be associated with better sprint and power performance in Caucasians. ACTN4 was initially identified in a screen for antigens to which antibodies strongly reacted in highly invasive breast cancer [7]. Honda reviews recent literature on the link between ACTN4 amplification, overexpression and spliced variants that predict metastatic potency in several types of tumors including breast, prostate, colon and lung [8]. He also summarizes evidence supporting a role of ACTN4 in transcriptional regulation by nuclear receptors and NF-κB and its link to tumorigenesis. Lastly, Honda suggests that ACTN4 may be a useful biomarker for evaluating treatment option in metastatic breast cancer. An elegant human genetics study has linked ACTN4 mutations to the kidney disorder focal segmental glomerular sclerosis (FSGS) [9]. This finding pioneered a novel concept that mutations in genes encoding cytoskeletal proteins in podocytes may be linked to glomerulopathy. Indeed, mutations of several cytoskeletal proteins have been subsequently found in patients with familial FSGS [10]. Interestingly, reduced expression of ACTN4 protein in kidney was also found in sporadic FSGS, minimal change disease and IgA nephropathy. Feng et al., thoroughly discusses the clinical presentation and biochemical and biophysical properties of disease causing ACTN4 mutations as well as related animal studies [11]. They further provide potential mechanisms underlying ACTN4-linked glomerulopathy. The physiological function of ACTN4 is unlikely limited to bundling actin and co-activating transcription. It is likely to also be a signaling molecule that transmits extracellular signalings to influence cell growth and differentiation. The intent of this thematic series is to summarize recent discoveries on actinin function and ACTN-related diseases and highlight future challenges in ACTN research.

The promyelocytic leukemia (PML) protein is a tumor suppressor originally identified in acute promyelocytic leukemia and implicated in tumorigenesis in multiple forms of cancer. PML protein is frequently down-regulated in various cancers, but PML mRNA levels are relatively similar between normal and cancerous tissues (1). These observations indicate that PML protein levels are tightly regulated, in part, through post-transcriptional modification. We previously demonstrated that the PML protein undergoes ubiquitination (Ub)-mediated degradation facilitated by an E3 ligase UHRF1 (2), and that SIRT1/SIRT5 promotes deacetylation and SUMO1 conjugation of PML (3) in response to oxidative stress. Here, we found that the small noncoding RNA miR-24 and miR-133 target 3' UTR of PML1 mRNA, the major PML isoform in primary human endothelial cells (ECs). In normal culture condition, miR-24 and miR-133 down-regulate PML1 protein expression in primary human ECs. However, miR-24 but not miR-133 up-regulates PML1 protein ...

Histone deacetylase 7 (HDAC7) is a member of class IIa HDACs that regulate myocyte enhancer factor-2 (MEF2)-mediated transcription and participate in multiple cellular processes such as T cell apoptosis. We have identified alpha-actinin 1 and 4 as class IIa HDAC-interacting proteins. The interaction domains are mapped to C terminus of alpha-actinin 4 and amino acids 72-172 of HDAC7. A point mutation in HDAC7 that disrupts its association with MEF2A also disrupts its association with alpha-actinin 4, indicating that MEF2A and alpha-actinin 4 binding sites largely overlap. We have also isolated a novel splice variant of alpha-actinin 4 that is predominantly localized in the nucleus, a pattern distinct from the full-length alpha-actinin 4, which is primarily distributed in the cytoplasm and plasma membrane. Using small interfering RNA, chromatin immunoprecipitation, and transient transfection assays, we show that alpha-actinin 4 potentiates expression of TAF55, a putative MEF2 target gene. Loss of MEF2A interaction correlates with loss of the ability of alpha-actinin 4 to potentiate TAF55 promoter activity. Ectopic expression of alpha-actinin 4, but not the mutant defective in MEF2A association, leads to disruption of HDAC7.MEF2A association and enhancement of MEF2-mediated transcription. Taken together, we have identified a novel mechanism by which HDAC7 activity is negatively regulated and uncovered a previously unknown function of alpha-actinin 4.

Retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers control gene expression through recruitment of co-repressors or co-activators, depending on their hormone binding status. We show that the helix 12 of RXRalpha and RARalpha is critical for recruitment of the co-regulators and transcriptional regulation by RXRalpha, RARalpha, and RXRalpha/RARalpha. LG268, an RXR-specific agonist, was able to promote co-activator association with the heterodimers, but was unable to dissociate co-repressors. Reconstitution experiments in yeast demonstrated that LG268 was capable of activating transcription by RXRalpha/RARalpha through recruitment of the co-activator. We hypothesize that the inability to release co-repressors from RXRalpha/RARalpha is responsible for the inability of LG268 to activate RXRalpha/RARalpha heterodimers in mammalian cells. Deletion of RARalpha helix 12 (RXRalpha/RARalpha Delta403) abolished both hormone-dependent dissociation from co-repressors and hormone-dependent association with co-activators. Deletion of RXRalpha helix 12 (RXRalpha Delta443/RARalpha) resulted in a higher binding affinity for co-repressors. Unexpectedly, RXRalpha Delta443/RARalpha also gained hormone-independent co-activator binding activity. Moreover, LG268 became an antagonist to RXRalpha Delta443/RARalpha heterodimers. These data suggest that the helix 12 of RXRalpha plays an inhibitory role in the recruitment of co-activators by unliganded RXRalpha/RARalpha.

Ataxin 1 (Atx1) is a foci-forming polyglutamine protein of unknown function, whose mutant form causes type 1 spinocerebellar ataxia in humans and exerts neurotoxicity in transgenic mouse and fly expressing mutant Atx1. In this study, we demonstrate that Atx1 interacts with the transcriptional corepressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) and with histone deacetylase 3. Atx1 binds chromosomes and mediates transcriptional repression when tethered to DNA. Interaction with SMRT-related factors is a conserved feature of Atx1, because Atx1 also binds SMRTER, a Drosophila cognate of SMRT. Significantly, mutant Atx1 forms aggregates in Drosophila , and such mutant Atx1-mediated aggregates sequester SMRTER. Consistently, the neurodegenerative eye phenotype caused by mutant Atx1 is enhanced by a Smrter mutation and, conversely, is suppressed by a chromosomal duplication that contains the wild type Smrter gene. Together, our results suggest that Atx1 is a transcriptional factor whose mutant form exerts its deleterious effects in part by perturbing corepressor-dependent transcriptional pathways.

Tip60 (Tat-interactive protein, 60 kDa), a cellular protein with intrinsic histone acetyltransferase activity, is involved in DNA damage repair and apoptosis. Recent studies have suggested that Tip60 acts either as a co-activator or a co-repressor to modulate transcription. In this study, we demonstrate that Tip60 represses reporter gene expression when it is fused to the Gal4 DNA binding domain. We also show that Tip60 associates with histone deacetylase 7 (HDAC7) through its N-terminal zinc finger-containing region and that HDAC7 activity is required for the repressive effect of Tip60. Because endogenous Tip60 interacts with STAT3, we hypothesized that Tip60 might complex with STAT3 and HDAC7 and modulate STAT3-mediated trans-activation. Consistent with this hypothesis, the overexpression of Tip60 represses STAT3-driven reporter gene expression, which can be further potentiated by the co-transfection of HDAC7. Furthermore, interleukin-9-induced c-myc expression, which depends on STAT3 activity, is abrogated by exogenous expression of Tip60. This is the first demonstration of which Tip60 represses STAT3 activity in part through the recruitment of HDAC7.

Metal matrix composites have been found to be quite useful in a number of engineering applications including the consumer goods. However, the machining of these materials is tough because of the abrasive characteristics of the reinforced particulates. In this paper drilling studies on Al2O3 aluminum-based metal matrix composites have been conducted by using different drills (high-speed steel, carbide-tipped, and polycrystalline diamond (PCD) drills) to produce holes in 10 and 20 vol.% (Al2O3)p/6061. The drilling forces were recorded using dynamometer. Surface finish was evaluated by a surface profilometer, and tool wear and geometry were inspected using optical and scanning electron microscopes (SEM). The drilling characteristics were evaluated in terms of drilling forces, tool wear, chip formation, and drilled-hole quality. It was found that PCD drills outperformed all other drills in terms of drilled-hole quality and minimum drilling forces induced. Better results achieved with the PCD drills are inline with other similar studies with other processes.

Acetylation of histone core particles plays an important role in modulating chromatin structure and gene expression. The acetylation status of the histone tails is determined by two opposing enzymatic activities, histone acetyltransferases and histone deacetylases (HDACs). Here we describe the isolation and characterization of HDAC10, a novel class II histone deacetylase. Molecular cloning and Northern blot analyses reveal that the HDAC10 transcript is widely expressed and subjected to alternative splicing. HDAC10 is both nuclear and cytoplasmic, a feature reminiscent of HDACs 4, 5, and 7. Distinct from other family members, HDAC10 harbors an amino-terminal catalytic domain and a carboxyl pseudo-repeat that shares significant homology with its catalytic domain. Mutational analysis reveals that transcriptional repression by HDAC10 requires its intrinsic histone deacetylase activity. Taken together, HDAC10 represents a distinct HDAC that may play a role in transcription regulation.

e23176 Background: Lactate considered a "waste product" lactate is a multifunctional modulator. Processes modulated by lactate are: 1. initiation of the transformation of the microenvironment 2.production of hyaluronan which interacts with RHAMM to cause cell motility, stabilize mitotic spindle, and supports stem cells. With these many processes, we hypothesized that lactate might interrupt the dormant state of the MOLT3 dormant cell line. Previous authors Indraccola et al, PNAS, 2006 reported that the dormant MOLT3 cell line implanted in SCID mice could be interrupted to become tumorigenic by co-implantation with VEGF,FGF, or irradiated (dead) Kaposi Sarcoma. We used this model to determine if lactate could stimulate tumorigenesis in a similar manner. Methods: In multiple cohorts of SCID mice, lactate was coimplanted with MOLT3 cells. In approximately 40 days, tumors grew rapidly in an exponential manner. Arterial perfusion was measured by contrast enhanced dual energy CT and MRI perfusion. Immunohistochemistry was performed for vascular stains CD21,VEGFR3, Notch 1, Ephrin B, VEGFA,VEGFD, Macrophages, VEGFD. Rna sequencing was performed on six samples, 3 Molt3 cell plugs, 3 MOLT3/lactate tumors (sequencing done at UCLA, data processed at Broad Street Institute MIT). Results: The data confirmed lactates's modulator role. Perfusion studies showed no arterial flow. The IHC showed many cancer cell with many mitotic figures and apoptosis.IHC confirmed presence of numerous macrophages, increased VEGFD, lymphatics and no arteries. Indraccola's reported their tumors showed no mitotic figures. 1114 genes were up/down regulated. In general rna showed: 1.mitochondrial dysfunction 2.Down regulated tumor suppressors 3) Up regulated oncogenes 4)Increased Stem cell markers 5) increased NFK-b and other pathways. Conclusions: Lactate induced an avascular "cancer" with features similar to natural pancreatic ductal and triple negative breast cancer. Results question the universality of the angiogenic switch. We hypothesize Lactate from marophages may be the cause of inflammation induced cancer.

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response.

Here, we describe the identification and characterization of a nuclear body (matrix-associated deacetylase body) whose formation and integrity depend on deacetylase activity. Typically, there are 20–40 0.5-μM bodies per nucleus, although the size and number can vary substantially. The structure appears to contain both class I and the recently described class II histone deacetylases (HDAC)5 and 7 along with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid receptor) and N-CoR (nuclear receptor corepressor). Addition of the deacetylase inhibitors trichostatin A and sodium butyrate completely disrupt these nuclear bodies, providing a demonstration that the integrity of a nuclear body is enzyme dependent. We demonstrate that HDAC5 and 7 can associate with at least 12 distinct proteins, including several members of the NuRD and Sin3A repression complexes, and appear to define a new but related complex.

Homeobox (hox) proteins have been shown to regulate cell fate and segment identity by promoting the expression of specific genetic programs. In contrast to their restricted biological action in vivo, however, most homeodomain factors exhibit promiscuous DNA binding properties in vitro, suggesting a requirement for additional cofactors that enhance target site selectivity. In this regard, the pbx family of homeobox genes has been found to heterodimerize with and thereby augment the DNA binding activity of certain hox proteins on a subset of potential target sites. Here we examine the transcriptional properties of a forced hox-pbx heterodimer containing the pancreas-specific orphan homeobox factor pdx fused to pbx-1a. Compared to the pdx monomer, the forced pdx-pbx1a dimer, displayed 10- to 20-fold-higher affinity for a consensus hox-pbx binding site but was completely unable to bind a hox monomer recognition site. The pdx-pbx dimer stimulated target gene expression via an N-terminal trans-activation domain in pdx that interacts with the coactivator CREB binding protein. The pdx-pbx dimer was also found to repress transcription via a C-terminal domain in pbx-1a that associates with the corepressors SMRT and NCoR. The transcriptional properties of the pdx-pbx1 complex appear to be regulated at the level of alternative splicing; a pdx-pbx polypeptide containing the pbx1b isoform, which lacks the C-terminal extension in pbx1a, was unable to repress target gene expression via NCoR-SMRT. Since pbx1a and pbx1b are differentially expressed in endocrine versus exocrine compartments of the adult pancreas, our results illustrate a novel mechanism by which pbx proteins may modulate the expression of specific genetic programs, either positively or negatively, during development.

The Drosophila ecdysone receptor (EcR)/ultraspiracle (USP) heterodimer is a key regulator in molting and metamorphoric processes, activating and repressing transcription in a sequence-specific manner. Here, we report the isolation of an EcR-interacting protein, SMRTER, which is structurally divergent but functionally similar to the vertebrate nuclear corepressors SMRT and N-CoR. SMRTER mediates repression by interacting with Sin3A, a repressor known to form a complex with the histone deacetylase Rpd3/HDAC. Importantly, we identify an EcR mutant allele that fails to bind SMRTER and is characterized by developmental defects and lethality. Together, these results reveal a novel nuclear receptor cofactor that exhibits evolutionary conservation in the mechanism to achieve repression and demonstrate the essential role of repression in hormone signaling.

Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase β-TrCP1 and a decrease in SMRT protein levels. We show that β-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of β-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced β-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.

The tumor suppressor promyelocytic leukemia protein (PML) predominantly resides in a structurally distinct sub-nuclear domain called PML nuclear bodies (NBs). Emerging evidences indicated that PML actively participates in many aspects of cellular processes, but the molecular mechanisms underlying PML regulation in response to stress and environmental cues are not complete. Post-translational modifications, such as sumoylation, phosphorylation, acetylation, and ubiquitination of PML add a complex layer of regulation to the physiological function of PML. In this review, we discuss the fast-moving horizon of post-translational modifications targeting PML.

Alpha-actinins (ACTNs) were originally identified as cytoskeletal proteins which cross-link filamentous actin to establish cytoskeletal architect that protects cells from mechanical stress and controls cell movement. Notably, unlike other ACTNs, alpha-actinin 4 (ACTN4) displays unique characteristics in signaling transduction, nuclear translocation, and gene expression regulation. Initial reports indicated that ACTN4 is part of the breast cancer cell motile apparatus and is highly expressed in the nucleus. These results imply that ACTN4 plays a role in breast cancer tumorigenesis. While several observations in breast cancer and other cancers support this hypothesis, little direct evidence links the tumorigenic phenotype with ACTN4-mediated pathological mechanisms. Recently, several studies have demonstrated that in addition to its role in coordinating cytoskeleton, ACTN4 interacts with signaling mediators, chromatin remodeling factors, and transcription factors including nuclear receptors. Thus, ACTN4 functions as a versatile promoter for breast cancer tumorigenesis and appears to be an ideal drug target for future therapeutic development.

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.