Person:

Slack, Frank

In C. elegans, intestinal autofluorescence (sometimes referred to as lipofuscin or “age pigment”) accumulates with age and is often used as a marker of health or the rate of aging. We show that this autofluorescent material is spectrally heterogeneous, and that materials that fluoresce under different excitation wavelengths have distinct biological properties. Red autofluorescence (visible with a TRITC filterset) correlates well with an individual's remaining days of life, and is therefore a candidate marker of health. In contrast, blue autofluorescence (via a DAPI filterset) is chiefly an indicator of an individual's incipient or recent demise. Thus, population averages of blue fluorescence essentially measure the fraction of dead or near-dead individuals. This is related to but distinct from the health of the living population. Green autofluorescence (via a FITC or GFP filterset) combines both properties, and is therefore ill suited as a marker of either death or health. Moreover, our results show that care must be taken to distinguish GFP expression near the time of death from full-body green autofluorescence. Finally, none of this autofluorescence increases after oxidative stress, suggesting that the material, or its biology in C. elegans, is distinct from lipofuscin as reported in the mammalian literature.

Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3′ untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3′ UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3′ UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3′ UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3′ UTR that is required for KRAS 3′ UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3′ UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3′ UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression.

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5′-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5′-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level.

The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions, and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function, and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual’s susceptibility to cancer.

In C. elegans, miRNAs are genetic biomarkers of aging. Similarly, multiple miRNAs are differentially expressed between younger and older persons, suggesting that miRNA-regulated biological mechanisms affecting aging are evolutionarily conserved. Previous human studies have not considered participants' lifespans, a key factor in identifying biomarkers of aging. Using PCR arrays, we measured miRNA levels from serum samples obtained longitudinally at ages 50, 55, and 60 from 16 non-Hispanic males who had documented lifespans from 58 to 92. Numerous miRNAs showed significant changes in expression levels. At age 50, 24 miRNAs were significantly upregulated, and 73 were significantly downregulated in the long-lived subgroup (76-92 years) as compared with the short-lived subgroup (58-75 years). In long-lived participants, the most upregulated was miR-373-5p, while the most downregulated was miR-15b-5p. Longitudinally, significant Pearson correlations were observed between lifespan and expression of nine miRNAs (p value<0.05). Six of these nine miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, 1225-3p) were also significantly up- or downregulated when comparing long-lived and short-lived participants. Twenty-four validated targets of these miRNAs encoded aging-associated proteins, including PARP1, IGF1R, and IGF2R. We propose that the expression profiles of the six miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, and 1225-3p) may be useful biomarkers of aging.

Elucidating the mechanisms involved in sensitizing radioresistant tumors to ionizing radiation (IR) treatments while minimizing injury to surrounding normal tissue is an important clinical goal. Due to their sequence-derived specificity and properties as gene regulators in IR-affected pathways, microRNAs (miRNAs) could serve as adjuvant therapeutic agents that alter cellular sensitivity to radiation treatment. To identify radiosensitizing miRNAs, we initially utilized the C. elegans vulval cell model, an in vivo system developed to study IR-dependent radiosensitivity as a measure of clonogenic cell death. We tested several candidate miRNA deletion mutants post γ-irradiation and identified cel-mir-237 as a miRNA which when deleted caused animals to be more resistant to IR, while cel-mir-237 overexpressing strains were IR-sensitive. Additionally, wild-type animals downregulated cel-mir-237 levels post IR in a time-dependent manner. We identified jun-1 (JUN transcription factor homolog) as a novel target of cel-mir-237. Specifically, jun-1 transcript levels increased in wild-type animals post-γ-irradiation, and loss of cel-mir-237 also resulted in higher jun-1 expression. As expected, loss of jun-1 resulted in IR sensitivity, similar to the phenotype of cel-mir-237 overexpressors. Since miR-237 is the homologue of human miR-125, we validated our findings in MCF-7 and MDA-MB-231 breast cancer cell lines, which harbor lower hsa-miR-125b levels than normal HMECs. Forced expression of hsa-miR-125b in these cells resulted in radiosensitivity, as seen by reduced clonogenic survival, enhanced apoptotic activity, and enhanced senescence post IR. Finally, re-expression of c-JUN in MDA-MB-231 cells promoted radio-resistance and abrogated miR-125-mediated radio-sensitization. Our findings suggest that overexpression of cel-mir-237 and its homologue, hsa-miR-125b, functions as sensitizers to γ-irradiation in both a nematode in vivo model and breast cancer cells, and could potentially be utilized as an adjuvant therapeutic to enhance radiation sensitivity.

The PD-L1/PD-1 pathway is a critical component of the immunosuppressive tumor microenvironment in acute myeloid leukemia (AML), but little is known about its regulation. We investigated the role of the MUC1 oncoprotein in modulating PD-L1 expression in AML. Silencing of MUC1 in AML cell lines suppressed PD-L1 expression without a decrease in PD-L1 mRNA levels, suggesting a post-transcriptional mechanism of regulation. We identified the microRNAs miR-200c and miR-34a as key regulators of PD-L1 expression in AML. Silencing of MUC1 in AML cells led to a marked increase in miR-200c and miR-34a levels, without changes in precursor microRNA, suggesting that MUC1 might regulate microRNA-processing. MUC1 signaling decreased the expression of the microRNA-processing protein DICER, via the suppression of c-Jun activity. NanoString (Seattle, WA, USA) array of MUC1-silenced AML cells demonstrated an increase in the majority of probed microRNAs. In an immunocompetent murine AML model, targeting of MUC1 led to a significant increase in leukemia-specific T cells. In concert, targeting MUC1 signaling in human AML cells resulted in enhanced sensitivity to T-cell-mediated lysis. These findings suggest MUC1 is a critical regulator of PD-L1 expression via its effects on microRNA levels and represents a potential therapeutic target to enhance anti-tumor immunity.

The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer.

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

The architecture of chromatin specifies eukaryotic cell identity by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase/peroxidase approach, integrative DNA And Protein Tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors, and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights with potential therapeutic implications. Our findings demonstrate the power of iDAPT as a discovery platform for both the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.