To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to a-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.

Purpose Severe skeletal muscle (SM) loss (sarcopenia), is associated with poor cancer outcomes including reduced survival and increased toxicity. This study investigates SM measures in metastatic breast cancer (MBC) patients receiving first line taxane-based chemotherapy and evaluates associations with treatment toxicity and other outcomes. Experimental Design Using computerized tomography (CT) images taken for the evaluation of disease burden, skeletal muscle area (SMA) and density (SMD) were measured at the 3rd lumbar vertebrae. Sarcopenia was defined as Skeletal Muscle Index (SMI=SMA/height2) ≤41. Skeletal Muscle Gauge (SMG) was created by multiplying SMI x SMD. Fisher’s exact tests, t-tests, the Kaplan-Meier method, and Cox regression modeling were used. Results MBC patients (N=40), median age 55 (rang 34–80), 58% sarcopenic, median SMG 1296 AU (SD 522). Grade 3–4 toxicity was found in 57% of sarcopenic vs 18% of non-sarcopenic patients (p=0.02). Toxicity-related hospitalizations were also higher in sarcopenic patients (39% vs 0%, p=0.005) as were any adverse events -- defined as any grade 3–4 toxicities, hospitalizations, dose reductions, or dose delay -- (74% vs 35%, p=0.02). Low SMG was associated with grade 3–4 toxicity (p=0.04), hospitalization (p=0.01) and time to treatment failure (for progression or toxicity) (p=0.03). Low SMG had a borderline significant association with any adverse event (p=0.06) and overall survival (p=0.07). Conclusions SM measures are associated with toxicity outcomes and survival in MBC patients receiving first line taxane-based chemotherapy. Further studies are needed to explore how routinely obtained CT scans can be used to individualize dosing and improve treatment planning.

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Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra-and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.