MCDB










microRNAs in development and disease
Frank Slack, Ph.D.

Frank Slack, Ph.D.

Professor of Molecular, Cellular & Developmental Biology
Yale University, KBT 716
PO Box 208103, 266 Whitney Ave
New Haven, CT 06520.
Phone (203) 432 3492; Fax (203) 432 6161
Email: frank.slack@yale.edu
Web site

B.Sc.(Hons.) University of Cape Town 1987; Ph.D. Tufts University School of Medicine 1993

Slack herniation
Micrograph of C. elegans with a mutation in the microRNA gene let-7. Adult animals herniate and burst through the vulva..

JR 1000
Wild-type animal expressing two green fluorescence protein (GFP) fusions that highlight the seam cell nuclei and junctions.

Worm GFP
Top. A C. elegans embryo expressing a green fluorescence protein (GFP) fusion in seam cells. Bottom. A DIC image of the same embryo

Gel
Black arrows show in vitro binding of the let-7 microRNA to the lin-41 3'UTR RNA containing let-7 complementary sites (lane 2). let-7 fails to bind to the same 3'UTR RNA missing those sites (lane 3). Grey arrows show let-7 binding to RNA containing just the let-7 complementary sites (lane 4). Free let-7 probe is in lane 1

Development, cancer and aging are intricately linked. Our lab focuses on using the advantages of C. elegans to find important genes and molecules that control aging and development of a stem cell pathway and testing if these genes are involved in aging, development and cancer in more complex organisms. A pathway of developmental timing genes, known as heterochronic genes has been identified through the genetic identification of C. elegans mutants that express stem cell fates either too early or too late relative to wild-type animals. This pathway controls the temporal progression of C. elegans development by regulating the abundance or activities of a succession of heterochronic genes over time, including key microRNAs. Since many of the C. elegans heterochronic genes and microRNAs control timing of cell differentiation and are related to human cancer genes, we are examining the role of their human homologues in cancer. We are also extrapolating our work in C. elegans to provide an understanding of how these genes and microRNAs regulate tissue differentiation and cell fate in mouse models. We are also developing microRNAs as cancer therapeutics. Lastly, since aging is the greatest risk factor for cancer, we are examining the roles of microRNAs in aging and longevity.

We are using molecular, genetic, bioinformatic and genomic approaches to understand:

  1. the role of microRNAs like lin-4 and let-7 in control of gene expression in development and cancer. lin-4 and let-7 are founding member of a large family of recently discovered microRNAs. These two ~20 nucleotide RNAs regulate gene expression in the heterochronic pathway. These RNAs bind to complementary sequences in the 3'UTR of their target gene mRNAs and through a mechanism that we are trying to understand, down-regulate their translation. We have isolated protein factors that bind to miRNAs and hope that their identity will shed light on the miRNA mechanism. We have described the minimal sequences necessary for miRNA control of target sequences and have used this information to design bioinformatics screens to identify novel miRNA targets, in an effort to understand how these miRNAs control differentiation. Most of the dozen or so targets we have identified encode transcription factors, leading to our assertion that these miRNAs are master temporal control genes. Another target is the C. elegans homologue of the human proto-oncogene RAS (see below). Both the lin-4 and let-7 miRNAs are transcriptionally regulated and begin to be expressed at critical times in development, just prior to the down-regulation of their targets. Developmental timing can therefore be distilled down to the timing of expression of these miRNAs – we are interested in what controls their transcription, as well as how they control the expression of their targets. We are also investigating the role of additional temporally regulated microRNAs during C. elegans development. Mis-regulation of genes that control cell proliferation and cell fate determination often contributes to cancer development. In C. elegans, let-7 controls the timing of proliferation versus differentiation decisionsby epidermal cells. In let-7 mutants, cells frequently fail to terminally differentiate, and instead elect to divide again, a hallmark of cancer. In C. elegans, let-7 directly regulates RAS, and another gene, lin-41, which is homologous to cancer genes, including PML, mutated in almost all cases of promylocytic leukemia. let-7 is conserved in humans, where we linked it to cancer. Specifically, human let-7 is poorly expressed or deleted in multiple cancers, including lung cancer, and over-expression of let-7 in lung cancer cells inhibits their growth, demonstrating a role for let-7 as a tumor suppressor in lung tissue. We have also shown that human let-7 is expressed in the lung and regulates the expression of important oncogenes implicated in lung cancer, including RAS. We are focusing on the role of let-7 in regulating proto-oncogene expression during lung development and cancer. We are developing anti-cancer drugs involving microRNAs.

  2. the temporal patterning role of the HBL-1 transcription factor, the LIN-28 RNA binding protein and the LIN-41 RING finger protein. By genetic arguments, hbl-1, lin-28 and lin-41 are major targets of the let-7 RNA, and there are let-7 complementary sequences in the 3'UTR of hbl-1, lin-28 and lin-41 that are responsive to let-7. While let-7 mutations lead to a reiteration of larval fates in the adult animal (in this case cells divide instead of terminally differentiate), hbl-1, lin-28 and lin-41 mutations display the opposite phenotype and precociously express adult fates in the larval animals (cells terminally differentiate instead of proliferate). hbl-1, lin-28 and lin-41 encode switches that must be tripped to progress from early fates to later fates. These proteins provide temporal cues that allow cells to decide whether to divide or terminally differentiate. lin-28 encodes a homologue of mammalian LIN28 oncogene, required as one of the stemness factor to reprogram differentiated cells to iPS cells. LIN-41 encodes a protein that belongs to a large super family that includes many human oncogenes and tumor suppressor genes. We are examining the mechanism of action of these factors in C. elegans andmouse knockouts. An emerging theme is of universal patterning mechanisms acting throughout the animal kingdom, and the C. elegans heterochronic pathway is emerging as an important guide to understanding stem cell biology in mammals.

  3. the role of the microRNAs in aging. lin-4 microRNA mutants and lin-14 mutants display defect in lifespan, that depend on genes in the insulin signaling pathway. We are currently investigating how these developmental timing genes influence timing of aging. In addition, we are discovering new microRNAs involved in aging in C. elegans and mouse. In addition, we discovered that the C. elegans homologue of Alzheimer's Precursor Protein functions in the developmental timing pathway. Thus we are investigating how these pathways interact during development and aging in C. elegans.


Recent Papers

Johnson, S., H. Grosshans, J. Shingar, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K. L. Reinert, D. Brown, and F. J. Slack. (2005) RAS is regulated by the let-7 microRNA family. Cell.120:635-647.

Boehm, M., and F. J. Slack. (2005). A developmental timing microRNA and its target regulate life span in C. elegans. Science. 310:1954-1957.

Banerjee, D., A. Kwok, S.-Y. Lin, and F. J. Slack. (2005) Developmental timing in C. elegans is regulated by kin-20 and tim-1, homologs of core circadian clock genes. Dev. Cell. 8:287-295.

Grosshans, H, T. Johnson, M. K. Reinert, Gerstein and F. J. Slack. The temporal patterning microRNA let-7 controls several transcription factors during the larval to adult transition in C. elegans. Dev. Cell. 8:321-330.

Esquela-Kerscher, A. and F. J. Slack. (2006) Oncomirs – microRNAs with a role in cancer. Nature Reviews Cancer. 6:259-269.

Johnson, C., Esquela-Kerscher, A., Stefani, G., Byrom, M., Kelnar, K., Ovcharenko, D., Wilson, M., Wang, X., Shelton, J., Shingara, J., Brown, D., and F.J. Slack. (2007) The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67:7713-7722.

Weidhaas, J., I. Babar, S. Naller, S. Roush, M. Boehm, and F. J. Slack. (2007) MicroRNAs as Potential Agents to Alter Resistance to Cytotoxic anti-Cancer Therapy. Cancer Res. 67:11111-11116.

Niwa, R. F. Zhou, C. Li and F. J. Slack. (2008) C. elegans Alzheimer APP-like is regulated by developmental timing microRNAs and their targets. Dev Biol. Jan 8; [Epub ahead of print]

Esquela-Kerscher, A., P. Trang, Cheng, A., Ford, L., Weidhaas, J, Brown, D. Bader, A, J. Weidhaas and F. J. Slack. (2008) The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle. 7:759-64

Lena J. Chin, Elena Ratner, Shuguang Leng, Rihong Zhai, Sunitha Nallur, Imran Babar, Roman-Ulrich Muller, Eva Straka, Li Su, Elizabeth A. Burki, Richard E. Crowell, Rajeshvari Patel, Trupti Kulkarni, Robert Homer, Daniel Zelterman, Kenneth K. Kidd, Yong Zhu, David C. Christiani, Steven A. Belinsky, Frank J. Slack and Joanne B. Weidhaas (2008) A SNP in a let-7 microRNA complementary site in the KRAS 3'UTR Increases Non Small Cell Lung Cancer Risk. Can. Res. 68:8535-40.

Schulman, B., X. Liang, C. Stahlhut, C. delConte, G. Stefani and F. J. Slack. (2008) The let-7 microRNA target, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle. Dec 13;7(24). [Epub ahead of print].

Kato, M., T. Paranjape, R. Ullrich, S. Nallur, E. Gillespie, K. Keane, A. Esquela-Kerscher, J. B. Weidhaas and F. J. Slack. (2009) The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human cells. Oncogene. 28:2419-24. Epub 2009 May 4.

Phong Trang, Pedro Medina, JF Wiggins, L Ruffino, K Kelnar, M Omotola, Robert Homer, David Brown, Andreas Bader, Joanne Weidhaas and F. J. Slack. (2009) Regression of murine lung tumors by the let-7 microRNA. Oncogene. Dec 7. [Epub ahead of print]

 

worms
Top. A field of mixed stage worms stained with an antibody to the heterochronic protein LIN-14. Only the larval stage 1 (L1) animals stain brightly. Bottom. The same field stained with DAPI to highlight nuclei.

MLin 41 mouse
Mouse embryos stained via in situ hybridization for mlin-41 expression. mlin-41 is seen in the limb buds, brachial arches and nervous system.

HBL 1 vrs WT

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