An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability.

INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE: Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.

Smad4 loss in mouse keratinocytes leads to increased susceptibility to UV carcinogenesis with reduced Ercc1-mediated DNA repair.

Smad4 loss occurs frequently in human skin squamous cell carcinoma (SCC), but it is unknown whether this loss increases UV-induced carcinogenesis, a major etiological factor in skin cancer. In the present study, mice with keratinocyte-specific Smad4 deletion (K14.Smad4(-/-)) and wild-type (WT) littermates were chronically UV-irradiated. Compared with WT, K14.Smad4(-/-) mice exhibited increased DNA damage and increased susceptibility to UV-induced skin cancer. Among genes involved in repairing UV-induced DNA damage, Excision repair cross-complementation group 1 (Ercc1) messenger RNA was significantly reduced in UV-treated K14.Smad4(-/-) skin compared with WT skin. Further analysis revealed that Smad4 loss confers reduced Snail binding to the Ercc1 regulatory elements, resulting in reduced Ercc1 transcription. Consistently, transient transfection of Snai1 into Smad4(-/-) keratinocytes led to increased repair of UV-induced DNA lesions. Transfection of Ercc1 into Smad4(-/-) keratinocytes restored repair of UV-induced DNA damage. Further, immunostaining revealed that the presence of Smad4 protein is associated with the presence of Snail and Ercc1 proteins in human skin SCC and precancerous actinic keratoses. Collectively, Smad4 loss-associated Snail reduction compromises Ercc1-mediated DNA repair, contributing to increased UV-induced skin carcinogenesis. Thus, we identified a role for Snail in UV-induced DNA repair.

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e.

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.

Cancer stem cells in head and neck cancer.

Head and neck cancer (HNC) is the sixth most common malignancy world-wide, however the survival rate has not improved for the past 20 years. In recent years, the cancer stem cell (CSC) hypothesis has gained ground in several malignancies and there is mounting evidence suggesting CSCs mediate tumor resistance to chemotherapy and radiation therapy. However, the CSC theory is also challenged at least in certain types of cancer. Here we review the progress of CSC studies in HNC, which suggest that HNC conforms to the CSC model. The identified CSC markers and their tumor initiation properties provide a framework for the development of novel therapeutic strategies for HNC.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance.

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

Opposing roles for Set2 and yFACT in regulating TBP binding at promoters.

Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16-Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

SWI/SNF binding to the HO promoter requires histone acetylation and stimulates TATA-binding protein recruitment.

We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.

Genetic interactions between Nhp6 and Gcn5 with Mot1 and the Ccr4-Not complex that regulate binding of TATA-binding protein in Saccharomyces cerevisiae.

Our previous work suggests that the Nhp6 HMGB protein stimulates RNA polymerase II transcription via the TATA-binding protein TBP and that Nhp6 functions in the same functional pathway as the Gcn5 histone acetyltransferase. In this report we examine the genetic relationship between Nhp6 and Gcn5 with the Mot1 and Ccr4-Not complexes, both of which have been implicated in regulating DNA binding by TBP. We find that combining either a nhp6ab or a gcn5 mutation with mot1, ccr4, not4, or not5 mutations results in lethality. Combining spt15 point mutations (in TBP) with either mot1 or ccr4 also results in either a growth defect or lethality. Several of these synthetic lethalities can be suppressed by overexpression of TFIIA, TBP, or Nhp6, suggesting that these genes facilitate formation of the TBP-TFIIA-DNA complex. The growth defect of a not5 mutant can be suppressed by a mot1 mutant. HO gene expression is reduced by nhp6ab, gcn5, or mot1 mutations, and the additive decreases in HO mRNA levels in nhp6ab mot1 and gcn5 mot1 strains suggest different modes of action. Chromatin immunoprecipitation experiments show decreased binding of TBP to promoters in mot1 mutants and a further decrease when combined with either nhp6ab or gcn5 mutations.